Influenza virus reassortment

ABSTRACT

New influenza donor strains for the production of reassortant influenza B viruses are provided.

This application claims the benefit of U.S. provisional application61/779,888 filed 13 Mar. 2013, the complete contents of which areincorporated herein by reference.

This invention was made in part with Government support under grant no.HHSO10020100061C awarded by the Biomedical Advanced Research andDevelopment Authority (BARDA). The Government has certain rights in theinvention.

TECHNICAL FIELD

This invention is in the field of influenza B virus reassortment.Furthermore, it relates to manufacturing vaccines for protecting againstinfluenza B viruses.

BACKGROUND ART

The most efficient protection against influenza infection is vaccinationagainst circulating strains and it is important to produce influenzaviruses for vaccine production as quickly as possible.

Wild-type influenza viruses often grow to low titres in eggs and cellculture. In order to obtain a better-growing virus strain for vaccineproduction it is possible to reassort the circulating vaccine strainwith a faster-growing high-yield donor strain. This can be achieved byco-infecting a culture host with the circulating influenza strain andthe high-yield donor strain and selecting for reassortant viruses whichcontain the hemagglutinin (HA) and neuraminidase (NA) segments from thevaccine strain and the other viral segments (i.e. those encoding PB1,PB2, PA, NP, M₁, M₂, NS₁ and NS₂) from the donor strain. Anotherapproach is to reassort the influenza viruses by reverse genetics (see,for example references 1 and 2).

Whilst it is common practice to use reassortant influenza A strains invaccine production, reassortant influenza B strains are not usually usedbecause wild-type influenza B viruses usually provide adequate yields ineggs. Furthermore, wild-type influenza B viruses have been reported tohave a growth advantage over reassortant influenza B viruses (see, forexample, reference 3). Accordingly, high growth influenza B reassortantshave been generated only for a small number of recent influenza Bviruses. These reassortants typically contain a mixture of backbone genesegments derived from B/Lee/40, B/Brisbane/60/08 and B/Panama/45/90 (4,5).

To date, only two reassortant influenza B viruses (BX-35 and BX-39) havebeen used for commercial vaccine manufacturing. BX-35 contains the HA,NA, PA, PB1, and NS segments from the B/Brisbane/60/08 strain, the PB2and M segments from B/Panama/45/90, and the NP segment from B/Lee/40.BX-39 contains the HA, NA, PBI, and M segments from the circulatingB/Hubei-Wujiagang/159/08 strain, the PA and NS segments fromB/Panama/45/90, and the PB2 and NP segments from B/Lee/40(6, 7).

There are currently only a limited number of donor strains forreassorting influenza B viruses for vaccine manufacture and the knownreassortant influenza B viruses do not always grow better than theparent strain. Thus, there is a need in the art to provide further andimproved donor strains for influenza B virus reassortment.

SUMMARY OF PREFERRED EMBODIMENTS

The invention thus provides reassortant influenza B viruses which cangrow at the same speed or faster in a culture host (particularly in cellculture) compared to the corresponding wild-type influenza B virus fromwhich the HA segment is derived. For example, the inventors havesurprisingly discovered that a reassortant influenza B virus whichcomprises the HA segment from a first influenza B virus and the NPand/or PB2 segment from a second influenza B virus which is aB/Victoria/2/87-like strain grows particularly well in cell culture andeggs. The B/Victoria/2/87-like strain may be B/Brisbane/60/08.

The invention also provides reassortant influenza B viruses comprisingthe HA segment from a first influenza B virus and the NP segment from asecond influenza B virus which is not B/Lee/40 or B/Ann Arbor/1/66 orB/Panama/45/90. For example, the reassortant influenza B virus may havea NP segment which does not have the sequence of SEQ ID NOs: 33, 38, 39or 43. The reassortant influenza B virus may also have a NP segmentwhich does not encode the protein of SEQ ID NOs: 19, 23, 44 or 45. Theinventors have discovered that reassortant influenza B viruses whichcomprise a NP segment from an influenza B virus other than B/Lee/40 orB/Ann Arbor/1/66 or B/Panama/45/90 can grow very well in a culture host.The reassortant influenza B virus may comprise both the NP and PB2segments from the second influenza B virus. The second influenza B virusis preferably a B/Victoria/2/87-like strain. The B/Victoria/2/87-likestrain may be B/Brisbane/60/08.

The inventors have also discovered that a reassortant influenza B viruscomprising the HA segment from a B/Yamagata/16/88-like strain and atleast one backbone segment from a B/Victoria/2/87-like strain can growwell in a culture host. The reassortant influenza B virus may comprisetwo, three, four, five or six backbone segments from theB/Victoria/2/87-like strain. In a preferred embodiment, the reassortantinfluenza B virus comprises all the backbone segments from theB/Victoria/2/87-like strain. The B/Victoria/2/87-like strain may beB/Brisbane/60/08.

The invention also provides a reassortant influenza B virus comprisingviral segments from a B/Victoria/2/87-like strain and aB/Yamagata/16/88-like strain, wherein the ratio of segments from theB/Victoria/2/87-like strain and the B/Yamagata/16/88-like strain is 1:7,2:6, 3:5, 4:4, 5:3, 6:2 or 7:1. A ratio of 7:1, 6:2, 4:4, 3:5 or 1:7, inparticular a ratio of 4:4, is preferred because such reassortantinfluenza B viruses grow particularly well in a culture host. TheB/Victoria/2/87-like strain may be B/Brisbane/60/08. TheB/Yamagata/16/88-like strain may be B/Panama/45/90. In theseembodiments, the reassortant influenza B virus usually does not compriseall backbone segments from the same influenza B donor strain.

Also provided is a reassortant influenza B virus which comprises:

a) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12,the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15; or

b) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 31,the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34; or

c) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 31,the PB2 segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15; or

d) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 12,the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15, or

e) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12,the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or

f) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31,the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 33, theNS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or

g) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31,the PB2 segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or

h) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12,the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 33, theNS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or

i) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12,the PB2 segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or

j) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 12,the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or

k) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31,the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, theNS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34.

In these reassortant influenza B viruses, the HA and NA segments may befrom any influenza B strain.

Reassortant influenza B viruses with the combinations of segments asdiscussed in section (a) to (k) above are preferred because theinventors have shown that they grow particularly well in culture hosts.The reassortant influenza B strains of sections (a), (b) and (e) growparticularly well in culture hosts and are therefore particularlypreferred.

The invention also provides variants of the reassortant influenza Bviruses identified in sections (a) to (k) above which comprise viralsegments that have at least 97% identity, at least 98% identity, or atleast 99% identity to the viral segments identified in these sections.Such variants can preferably grow to a viral titre in a culture hostwhich is within 3% of the viral titre achieved with the reassortantinfluenza B strain from which the variant is derived in the same timeand under the same growth conditions.

The invention provides methods of preparing the reassortant influenza Bviruses of the invention. These methods comprise steps of (i)introducing into a culture host one or more expression construct(s)which encode(s) the viral segments required to produce a reassortantinfluenza B virus of the invention and (ii) culturing the culture hostin order to produce the reassortant virus; and optionally (iii)purifying the virus obtained in step (ii).

These methods may further comprise steps of: (iv) infecting a culturehost with the virus obtained in step (ii) or step (iii); (v) culturingthe culture host from step (iv) to produce further virus; and optionally(vi) purifying the virus obtained in step (v).

Expression constructs which can be used in the methods of the inventionare also provided.

For example, the expression construct(s) may encode a reassortantinfluenza B virus comprising the HA segment from a first influenza Bvirus and the NP and/or PB2 segment from a second influenza B viruswhich is a B/Victoria/2/87-like strain. The NP and PB2 segments may bothbe from the B/Victoria/2/87-like strain. The B/Victoria/2/87-like strainis preferably B/Brisbane/60/08.

The expression construct(s) may also encode a reassortant influenza Bvirus comprising the HA segment from a first influenza B virus and theNP segment from a second influenza B virus which is not B/Lee/40 orB/Ann Arbor/1/66 or B/Panama/45/90. For example, the expressionconstruct(s) may not encode a NP segment with the sequence of SEQ IDNOs: 19, 23, 44 or 45. The NP and PB2 segments may both be from thesecond influenza B virus. The second influenza B virus may be aB/Victoria/2/87-like strain and is preferably B/Brisbane/60/08.

The “first influenza virus” and the “second influenza virus” aredifferent to each other.

The expression construct(s) can encode a reassortant influenza B viruscomprising viral segments from a B/Victoria/2/87-like strain and aB/Yamagata/16/88-like strain, wherein the ratio of segments from theB/Victoria/2/87-like strain and the B/Yamagata/16/88-like strain is 1:7,2:6, 3:5, 4:4, 5:3, 6:2 or 7:1. A ratio of 7:1, 6:2, 4:4, 3:5 or 1:7, inparticular a ratio of 4:4, is preferred. The B/Victoria/2/87-like strainmay be B/Brisbane/60/08. The B/Yamagata/16/88-like strain may beB/Panama/45/90.

Also provided are expression construct(s) which encode(s) a reassortantinfluenza B virus as described above.

The invention provides an expression system comprising one or moreexpression construct(s) of the invention. The invention also provides ahost cell comprising an expression system of the invention. These hostcells can express an influenza B virus from the expression construct(s)in the expression system.

The invention also provides a method for producing influenza virusescomprising steps of (a) infecting a culture host with a reassortantvirus of the invention; (b) culturing the host from step (a) to producethe virus; and optionally (c) purifying the virus obtained in step (b).

The invention also provides a method of preparing a vaccine, comprisingthe steps of (a) preparing a virus by the methods of any one of theembodiments described above and (b) preparing a vaccine from the virus.

Also provided is a method of preparing a vaccine from the reassortantinfluenza B virus of the invention.

The invention also provides a vaccine which can be obtained by themethods of the invention.

Reassortant Viruses

The reassortant influenza B strains of the invention contain viralsegments from a vaccine strain and one or more donor strain(s). Thevaccine strain is the influenza strain which provides the HA segment ofthe reassortant influenza B strain. The vaccine strain can be any strainand can vary from season to season.

A donor strain is an influenza B strain which provides one or more ofthe backbone segments (i.e. those encoding PB1, PB2, PA, NP, M₁, M₂, NS₁and NS₂) of the influenza B strain. The NA segment may also be providedby a donor strain or it may be provided by the vaccine strain. Thereassortant influenza B viruses of the invention may also comprise oneor more, but not all, of the backbone segments from the vaccine strain.As the reassortant influenza B virus contains a total of eight segments,it will therefore contain x (wherein x is from 1-7) viral segments fromthe vaccine strain and 8-x viral segments from the one or more donorstrain(s).

As mentioned above, the purpose of the invention is to providereassortant influenza B strains which, once rescued, can grow to higheror similar viral titres in a culture host. Thus, the reassortantinfluenza B virus strains of the invention can grow to higher or similarviral titres in cell culture and/or in eggs in the same time (forexample 12 hours, 24 hours, 48 hours or 72 hours) and under the samegrowth conditions compared to the wild-type vaccine strain. Inparticular, they can grow to higher or similar viral titres in MDCKcells (such as MDCK 33016) in the same time and under the same growthconditions compared to the wild-type vaccine strain. The viral titre canbe determined by standard methods known to those of skill in the art.Usefully, the reassortant influenza B viruses of the invention mayachieve a viral titre which is at least 5% higher, at least 10% higher,at least 20% higher, at least 50% higher, at least 100% higher, at least200% higher, or at least 500% higher than the viral titre of thewild-type vaccine strain in the same time frame and under the sameconditions. The reassortant influenza B viruses may also grow to similarviral titres in the same time and under the same growth conditionscompared to the wild-type vaccine strain. A similar titre in thiscontext means that the reassortant influenza B viruses grow to a titrewhich is within 3% of the viral titre achieved with the wild-typevaccine strain in the same time and under the same growth conditions(i.e. wild-type titre ±3%).

Influenza B viruses currently do not display different HA subtypes, butinfluenza B virus strains do fall into two distinct lineages. Theselineages emerged in the late 1980s and have HAs which can beantigenically and/or genetically distinguished from each other [8].Current influenza B virus strains are either B/Victoria/2/87-like orB/Yamagata/16/88-like. These strains are usually distinguishedantigenically, but differences in amino acid sequences have also beendescribed for distinguishing the two lineages e.g. B/Yamagata/16/88-likestrains often (but not always) have HA proteins with deletions at aminoacid residue 164, numbered relative to the ‘Lee40’ HA sequence [9]. Insome embodiments, the reassortant influenza B viruses of the inventionmay comprise viral segments from a B/Victoria/2/87-like strain. They maycomprise viral segments from a B/Yamagata/16/88-like strain.Alternatively, they may comprise viral segments from aB/Victoria/2/87-like strain and a B/Yamagata/16/88-like strain.

Where the reassortant influenza B virus comprises viral segments fromtwo or more influenza B virus strains, these viral segments may bederived from influenza B strains which have related neuraminidases. Forinstance, the influenza B strains which provide the viral segments mayboth have a B/Victoria/2/87-like neuraminidase [10] or may both have aB/Yamagata/16/88-like neuraminidase. For example, twoB/Victoria/2/87-like neuraminidases may both have one or more of thefollowing sequence characteristics: (1) not a serine at residue 27, butpreferably a leucine; (2) not a glutamate at residue 44, but preferablya lysine; (3) not a threonine at residue 46, but preferably anisoleucine; (4) not a proline at residue 51, but preferably a serine;(5) not an arginine at residue 65, but preferably a histidine; (6) not aglycine at residue 70, but preferably a glutamate; (7) not a leucine atresidue 73, but preferably a phenylalanine; and/or (8) not a proline atresidue 88, but preferably a glutamine. Similarly, in some embodimentsthe neuraminidase may have a deletion at residue 43, or it may have athreonine; a deletion at residue 43, arising from a trinucleotidedeletion in the NA gene, which has been reported as a characteristic ofB/Victoria/2/87-like strains, although recent strains have regainedThr-43 [10]. Conversely, of course, the opposite characteristics may beshared by two B/Yamagata/16/88-like neuraminidases e.g. S27, E44, T46,P51, R65, G70, L73, and/or P88. These amino acids are numbered relativeto the ‘Lee40’ neuraminidase sequence [11]. The reassortant influenza Bvirus may comprise a NA segment with the characteristics describedabove. Alternatively, or in addition, the reassortant influenza B virusmay comprise a viral segment (other than NA) from an influenza B strainwith a NA segment with the characteristics described above.

The backbone viral segments of an influenza B virus which is aB/Victoria/2/87-like strain can have a higher level of identity to thecorresponding viral segment from B/Victoria/2/87 than it does to thecorresponding viral segment of B/Yamagata/16/88 and vice versa. Forexample, the NP segment of B/Panama/45/90 (which is aB/Yamagata/16/88-like strain) has 99% identity to the NP segment ofB/Yamagata/16/88 and only 96% identity to the NP segment ofB/Victoria/2/87.

Where the reassortant influenza B virus of the invention comprises abackbone viral segment from a B/Victoria/2/87-like strain, the viralsegments may encode proteins with the following sequences. The PAprotein may have at least 97% identity, at least 98%, at least 99%identity or 100% identity to the sequence of SEQ ID NO: 1. The PB1protein may have at least 97% identity, at least 98%, at least 99%identity or 100% identity to the sequence of SEQ ID NO: 2. The PB2protein may have at least 97%, at least 98%, at least 99% or 100%identity to the sequence of SEQ ID NO: 3. The NP protein may have atleast 97% identity, at least 98%, at least 99% identity or 100% identityto the sequence of SEQ ID NO: 4. The M₁ protein may have at least 97%identity, at least 98%, at least 99% identity or 100% identity to thesequence of SEQ ID NO: 5. The M₂ protein may have at least 97% identity,at least 98%, at least 99% identity or 100% identity to the sequence ofSEQ ID NO: 6. The NS₁ protein may have at least 97% identity, at least98%, at least 99% identity or 100% identity to the sequence of SEQ IDNO: 7. The NS₂ protein may have at least 97% identity, at least 98%, atleast 99% identity or 100% identity to the sequence of SEQ ID NO: 8. Insome embodiments, the reassortant influenza B virus may also compriseall of these backbone segments.

Where the reassortant influenza B viruses of the invention comprise abackbone viral segment from a B/Yamagata/16/88-like strain, the viralsegment may encode proteins with the following sequences. The PA proteinmay have at least 97% identity, at least 98%, at least 99% identity or100% identity to the sequence of SEQ ID NO: 20. The PB1 protein may haveat least 97% identity, at least 98%, at least 99% identity or 100%identity to the sequence of SEQ ID NO: 21. The PB2 protein may have atleast 97%, at least 98%, at least 99% or 100% identity to the sequenceof SEQ ID NO: 22. The NP protein may have at least 97% identity, atleast 98%, at least 99% identity or 100% identity to the sequence of SEQID NO: 23. The M₁ protein may have at least 97% identity, at least 98%,at least 99% identity or 100% identity to the sequence of SEQ ID NO: 24.The M₂ protein may have at least 97% identity, at least 98%, at least99% identity or 100% identity to the sequence of SEQ ID NO: 25. The NS₁protein may have at least 97% identity, at least 98%, at least 99%identity or 100% identity to the sequence of SEQ ID NO: 26. The NS₂protein may have at least 97% identity, at least 98%, at least 99%identity or 100% identity to the sequence of SEQ ID NO: 27.

The invention can be practised with donor strains having a viral segmentthat has at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95% or at leastabout 99%, or 100% identity to a sequence of SEQ ID NOs 11-16 or 30-35.Due to the degeneracy of the genetic code, it is possible to have thesame polypeptide encoded by several nucleic acids with differentsequences. For example, the nucleic acid sequences of SEQ ID NOs: 40 and41 have only 73% identity even though they encode the same viralprotein. Thus, the invention may be practised with viral segments thatencode the same polypeptides as the sequences of SEQ ID NOs 11-16 or30-35.

In general a reassortant influenza virus will contain only one of eachbackbone segment. For example, when the influenza virus comprises the NPsegment from B/Brisbane/60/08 it will not at the same time comprise theNP segment from another influenza strain.

In some embodiments, the reassortant influenza B virus of the inventionmay comprise all backbone segments from the same influenza B donorstrain. It may alternatively comprise backbone segments from more thanone influenza donor strain, for example from two, three, four or fivedonor strains. Where the reassortant influenza B virus comprisesbackbone segments from two or three donor strains, each donor strain mayprovide more than one of the backbone segments of the reassortantinfluenza B virus, but one or two of the donor strains can also provideonly a single backbone segment. It is preferred that at least one of thebackbone segments is from a B/Yamagata/16/88-like strain as theinventors have found that such reassortant influenza viruses grow wellin cell culture. A preferred B/Yamagata/16/88-like strain in the contextof the invention is B/Panama/45/90. In general the reassortant influenzaB virus cannot comprise more than six backbone segments. Accordingly,for example, if one of the donor strains provides five of the viralsegments, the reassortant influenza B virus can only comprise backbonesegments from a total of two different influenza strains (for example,two donor strains or a donor strain and a vaccine strain).

When the reassortant influenza B virus comprises the backbone segmentsfrom a single donor strain, the reassortant viruses will generallyinclude segments from the donor strain and the vaccine strain in a ratioof 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7:1. When the reassortant virusescomprise backbone segments from two donor strains, the reassortant viruscan include segments from the first donor strain, the second donorstrain and the vaccine strain in a ratio of 1:1:6, 1:2:5, 1:3:4, 1:4:3,1:5:2, 1:6:1, 2:1:5, 2:2:4, 2:3:3, 2:4:2, 2:5:1, 3:1:4, 3:2:3, 3:3:2,3:4:1, 4:1:3, 4:2:2, 4:3:1, 5:1:2, 5:2:1 or 6:1:1.

The reassortant influenza B viruses contain the HA segment from thevaccine strain as this encodes the main vaccine antigens of theinfluenza virus and therefore comes from the vaccine strain. Thereassortant viruses of the invention preferably also have the NA segmentfrom the vaccine strain, but the invention also encompasses reassortantswhich comprise the HA and NA segments from different strains.

Strains which can be used as vaccine strains include strains which areresistant to antiviral therapy (e.g. resistant to oseltamivir [12]and/or zanamivir), including resistant pandemic strains [13].

Reassortant viruses which contain an NS segment that does not encode afunctional NS protein are also within the scope of the presentinvention. NS1 knockout mutants are described in reference 14. TheseNS1-mutant virus strains are particularly suitable for preparing liveattenuated influenza vaccines.

Variations in the DNA and the amino acid sequence may stem fromspontaneous mutations which can occur during passaging of the viruses.Such variant influenza strains can also be used in the invention.

Reverse Genetics

The invention is particularly suitable for producing the reassortantinfluenza B virus strains through reverse genetics techniques. In thesetechniques, the viruses are produced in culture hosts using one or moreexpression construct(s). The expression construct(s) may encode all thesegments which are necessary to produce the reassortant influenza Bviruses of the invention.

Reverse genetics for influenza viruses can be practised with 12 plasmidsto express the four proteins required to initiate replication andtranscription (PB1, PB2, PA and NP) and all eight viral genome segments.To reduce the number of constructs, however, a plurality of RNApolymerase I transcription cassettes (for viral RNA synthesis) can beincluded on a single plasmid (e.g. sequences encoding 1, 2, 3, 4, 5, 6,7 or all 8 influenza vRNA segments), and a plurality of protein-codingregions with RNA polymerase II promoters on another plasmid (e.g.sequences encoding 1, 2, 3, 4, 5, 6, 7 or 8 influenza mRNA transcripts)[15]. It is also possible to include one or more influenza vRNA segmentsunder control of a pol I promoter and one or more influenza proteincoding regions under control of another promoter, in particular a pol IIpromoter, on the same plasmid. This is preferably done by usingbi-directional plasmids.

Preferred aspects of the reference 15 method involve: (a) PB1, PB2, NPand PA mRNA-encoding regions on a single expression construct; and (b)all 8 vRNA encoding segments on a single expression construct. Includingthe neuraminidase (NA) and hemagglutinin (HA) segments on one expressionconstruct and the six other viral segments on another expressionconstruct is particularly preferred as newly emerging influenza virusstrains usually have mutations in the NA and/or HA segments.

Therefore, the advantage of having the HA and/or NA segments on aseparate expression construct is that only the vector comprising the HAand NA sequence needs to be replaced. Thus, in one aspect of theinvention the NA and/or HA segments of the vaccine strain may beincluded on one expression construct and the vRNA encoding segments fromthe donor strain(s) of the invention, excluding the HA and/or NAsegment(s), are included on a different expression construct. Theinvention thus provides an expression construct comprising one, two,three, four, five or six vRNA encoding backbone viral segments of adonor strain of the invention. The expression construct may not compriseHA and/or NA viral segments that produce a functional HA and/or NAprotein.

Known reverse genetics systems involve expressing DNA molecules whichencode desired viral RNA (vRNA) molecules from pol I promoters,bacterial RNA polymerase promoters, bacteriophage polymerase promoters,etc. As influenza viruses require the presence of viral polymerase toinitiate the life cycle, systems may also provide these proteins e.g.the system further comprises DNA molecules that encode viral polymeraseproteins such that expression of both types of DNA leads to assembly ofa complete infectious virus. It is also possible to supply the viralpolymerase as a protein.

Where reverse genetics is used for the expression of influenza vRNA, itwill be evident to the person skilled in the art that precise spacing ofthe sequence elements with reference to each other is important for thepolymerase to initiate replication. It is therefore important that theDNA molecule encoding the viral RNA is positioned correctly between thepol I promoter and the termination sequence, but this positioning iswell within the capabilities of those who work with reverse geneticssystems.

In order to produce a recombinant virus, a cell must express allsegments of the viral genome which are necessary to assemble a virion.DNA cloned into the expression constructs of the present inventionpreferably provides all of the viral RNA and proteins, but it is alsopossible to use a helper virus to provide some of the RNA and proteins,although systems which do not use a helper virus are preferred. As theinfluenza virus is a segmented virus, the viral genome will usually beexpressed using more than one expression construct in the methods of theinvention. It is also envisioned, however, to combine one or moresegments or even all segments of the viral genome on a single expressionconstruct.

In some embodiments an expression construct will also be included whichleads to expression of an accessory protein in the host cell. Forinstance, it can be advantageous to express a non-viral serine protease(e.g. trypsin) as part of a reverse genetics system.

Expression Constructs

Expression constructs used in the expression systems of the inventionmay be uni-directional or bi-directional expression constructs. Wheremore than one transgene is used in the methods (whether on the same ordifferent expression constructs) it is possible to use uni-directionaland/or bi-directional expression.

As influenza viruses require a protein for infectivity, it is generallypreferred to use bi-directional expression constructs as this reducesthe total number of expression constructs required by the host cell.Thus, the method of the invention may utilise at least onebi-directional expression construct wherein a gene or cDNA is locatedbetween an upstream pol II promoter and a downstream non-endogenous polI promoter. Transcription of the gene or cDNA from the pol II promoterproduces capped positive-sense viral mRNA which can be translated into aprotein, while transcription from the non-endogenous pol I promoterproduces negative-sense vRNA. The bi-directional expression constructmay be a bi-directional expression vector.

Bi-directional expression constructs contain at least two promoterswhich drive expression in different directions (i.e. both 5′ to 3′ and3′ to 5′) from the same construct. The two promoters can be operablylinked to different strands of the same double stranded DNA. Preferably,one of the promoters is a pol I promoter and at least one of the otherpromoters is a pol II promoter. This is useful as the pol I promoter canbe used to express uncapped vRNAs while the pol II promoter can be usedto transcribe mRNAs which can subsequently be translated into proteins,thus allowing simultaneous expression of RNA and protein from the sameconstruct. Where more than one expression construct is used within anexpression system, the promoters may be a mixture of endogenous andnon-endogenous promoters.

The pol I and pol II promoters used in the expression constructs may beendogenous to an organism from the same taxonomic order from which thehost cell is derived. Alternatively, the promoters can be derived froman organism in a different taxonomic order than the host cell. The term“order” refers to conventional taxonomic ranking, and examples of ordersare primates, rodentia, carnivora, marsupialia, cetacean, etc. Humansand chimpanzees are in the same taxonomic order (primates), but humansand dogs are in different orders (primates vs. carnivora). For example,the human pol I promoter can be used to express viral segments in caninecells (e.g. MDCK cells) [16].

The expression construct will typically include an RNA transcriptiontermination sequence. The termination sequence may be an endogenoustermination sequence or a termination sequence which is not endogenousto the host cell. Suitable termination sequences will be evident tothose of skill in the art and include, but are not limited to, RNApolymerase I transcription termination sequence, RNA polymerase IItranscription termination sequence, and ribozymes. Furthermore, theexpression constructs may contain one or more polyadenylation signalsfor mRNAs, particularly at the end of a gene whose expression iscontrolled by a pol II promoter.

An expression system may contain at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten, at least eleven or at least twelve expressionconstructs.

An expression construct may be a vector, such as a plasmid or otherepisomal construct. Such vectors will typically comprise at least onebacterial and/or eukaryotic origin of replication. Furthermore, thevector may comprise a selectable marker which allows for selection inprokaryotic and/or eukaryotic cells. Examples of such selectable markersare genes conferring resistance to antibiotics, such as ampicillin orkanamycin. The vector may further comprise one or more multiple cloningsites to facilitate cloning of a DNA sequence.

As an alternative, an expression construct may be a linear expressionconstruct. Such linear expression constructs will typically not containany amplification and/or selection sequences. However, linear constructscomprising such amplification and/or selection sequences are also withinthe scope of the present invention. Reference 17 describes individuallinear expression constructs for each viral segment. It is also possibleto include more than one, for example two, three four, five or six viralsegments on the same linear expression construct. Such a system has beendescribed, for example, in reference 18. It is also possible to use anexpression system in which some viral segments (for example the HAand/or NA segment) are encoded on a linear construct and the remainingviral segments (for example the backbone segments) are encoded on anon-linear construct, such as a vector, a plasmid or other episomalconstruct.

Expression constructs can be generated using methods known in the art.Such methods were described, for example, in reference 19. Where theexpression construct is a linear expression construct, it is possible tolinearise it before introduction into the host cell utilising a singlerestriction enzyme site. Alternatively, it is possible to excise theexpression construct from a vector using at least two restriction enzymesites. Furthermore, it is also possible to obtain a linear expressionconstruct by amplifying it using a nucleic acid amplification technique(e.g. by PCR).

The expression constructs used in the systems of the invention may benon-bacterial expression constructs. This means that the construct candrive expression in a eukaryotic cell of viral RNA segments encodedtherein, but it does not include components which would be required forpropagation of the construct in bacteria. Thus the construct will notinclude a bacterial origin of replication (ori), and usually will notinclude a bacterial selection marker (e.g. an antibiotic resistancemarker). Such expression constructs are described in reference 20.

The expression constructs may be prepared by chemical synthesis. Theexpression constructs may either be prepared entirely by chemicalsynthesis or in part. Suitable methods for preparing expressionconstructs by chemical synthesis are described, for example, inreference 20.

The expression constructs of the invention can be introduced into hostcells using any technique known to those of skill in the art. Forexample, expression constructs can be introduced into host cells byemploying electroporation, DEAE-dextran, calcium phosphateprecipitation, liposomes, microinjection, or microparticle-bombardment.The expression construct(s) can be introduced into the same cell typewhich is subsequently used for the propagation of the reassortantinfluenza B viruses. Alternatively, the cells into which the expressionconstructs are introduced and the cells used for propagation of thereassortant influenza B viruses may be different. In some embodiments,untransfected cells of the same or a different cell type may be added tothe host cells following transfection of the host cells with theexpression construct(s), as described in reference 21.

Conventional Reassortment

Traditionally, influenza viruses are reassorted by co-infecting aculture host, usually eggs, with a donor strain and a vaccine strain.Reassortant viruses are selected by adding antibodies with specificityfor the HA and/or NA proteins of the donor strain in order to select forreassortant viruses that contain the vaccine strain's HA and/or NAproteins. Over several passages of this treatment one can select forfast growing reassortant viruses containing the vaccine strain's HAand/or NA segments.

The reassortant influenza viruses can also be selected by adding aninhibitory agent which preferentially reduces the transcription and/ortranslation of the viral segments are not present in the desiredreassortant influenza virus, as taught in WO2011/145081.

The invention is suitable for use in these methods. It can be easier touse a vaccine strain from a different influenza B lineage compared tothe donor strain(s) as this facilitates selection for reassortantviruses. It is also possible, however, to use a vaccine strain from thesame influenza B lineage as the donor strain(s) and in some aspects ofthe invention this preferred. In this case, antibodies or inhibitoryagents with preferential specificity for the HA and/or NA proteins ofthe donor strain(s) should be available.

Culture Host

The culture host for use in the invention, can be any eukaryotic cellthat can produce the virus of interest. The invention will typically usea cell line although, for example, primary cells may be used as analternative. The cell will typically be mammalian or avian. Suitablemammalian cells include, but are not limited to, hamster, cattle,primate (including humans and monkeys) and dog cells. Various cell typesmay be used, such as kidney cells, fibroblasts, retinal cells, lungcells, etc. Examples of suitable hamster cells are the cell lines havingthe names BHK21 or HKCC. Suitable monkey cells are e.g. African greenmonkey cells, such as kidney cells as in the Vero cell line [22-24].Suitable dog cells are e.g. kidney cells, as in the CLDK and MDCK celllines. Suitable avian embryonic stem cells, include the EBx cell linederived from chicken embryonic stem cells, EB45, EB14, and EB14-074[25]. Chicken embryo fibroblasts (CEF) may also be used.

Further suitable cells include, but are not limited to: CHO; 293T; BHK;MRC 5; PER.C6 [26]; FRhL2; WI-38; etc. Suitable cells are widelyavailable e.g. from the American Type Cell Culture (ATCC) collection[27], from the Coriell Cell Repositories [28], or from the EuropeanCollection of Cell

Cultures (ECACC). For example, the ATCC supplies various different Verocells under catalogue numbers CCL 81, CCL 81.2, CRL 1586 and CRL-1587,and it supplies MDCK cells under catalogue number CCL 34. PER.C6 isavailable from the ECACC under deposit number 96022940.

Preferred cells for use in the invention are MDCK cells [29-31], derivedfrom Madin Darby canine kidney. The original MDCK cells are availablefrom the ATCC as CCL 34. It is preferred that derivatives of MDCK cellsare used. Such derivatives were described, for instance, in reference 29which discloses MDCK cells that were adapted for growth in suspensionculture (‘MDCK 33016’ or ‘33016-PF’, deposited as DSM ACC 2219).Furthermore, reference 32 discloses MDCK-derived cells that grow insuspension in serum free culture (‘B-702’, deposited as FERM BP-7449).In some embodiments, the MDCK cell line used may be tumorigenic. It isalso envisioned to use non-tumorigenic MDCK cells. For example,reference 33 discloses non tumorigenic MDCK cells, including ‘MDCK-S’(ATCC PTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501), ‘MDCK-SF102’ (ATCCPTA-6502) and ‘MDCK-SF103’ (ATCC PTA-6503). Reference 34 discloses MDCKcells with high susceptibility to infection, including ‘MDCK.5F1’ cells(ATCC CRL 12042).

It is possible to use a mixture of more than one cell type to practisethe methods of the invention. However, it is preferred that the methodsof the invention are practised with a single cell type e.g. withmonoclonal cells. Preferably, the cells used in the methods of thepresent invention are from a single cell line. Furthermore, the samecell line may be used for reassorting the virus and for any subsequentpropagation of the virus.

Preferably, the cells are cultured in the absence of serum, to avoid acommon source of contaminants. Various serum-free media for eukaryoticcell culture are known to the person skilled in the art (e.g. Iscove'smedium, ultra CHO medium (BioWhittaker), EX-CELL (JRH Biosciences)).Furthermore, protein-free media may be used (e.g. PF-CHO (JRHBiosciences)). Otherwise, the cells for replication can also be culturedin the customary serum-containing media (e.g. MEM or DMEM medium with0.5% to 10% of fetal calf serum).

The cells may be in adherent culture or in suspension.

The reassortant influenza B viruses of the invention may also bepropagated using eggs as the culture host. The current standard methodfor influenza virus growth for vaccines uses embryonated hen eggs, withvirus being purified from the egg contents (allantoic fluid). It is alsopossible to passage a virus through eggs and subsequently propagate itin cell culture and vice versa.

Virus Preparation

In one embodiment, the invention provides a method for producinginfluenza viruses comprising steps of (a) infecting a culture host witha reassortant virus of the invention; (b) culturing the host from step(a) to produce the virus; and optionally (c) purifying the virusproduced in step (b).

The culture host in step (b) may be cells (as described above) orembryonated hen eggs. Where cells are used as a culture host in thisaspect of the invention, it is known that cell culture conditions (e.g.temperature, cell density, pH value, etc.) are variable over a widerange subject to the cell line and the virus employed and can be adaptedto the requirements of the application. The following informationtherefore merely represents guidelines.

Cells are preferably cultured in serum-free or protein-free media.

Multiplication of the cells can be conducted in accordance with methodsknown to those of skill in the art. For example, the cells can becultivated in a perfusion system using ordinary support methods likecentrifugation or filtration. Moreover, the cells can be multipliedaccording to the invention in a fed-batch system before infection. Inthe context of the present invention, a culture system is referred to asa fed-batch system in which the cells are initially cultured in a batchsystem and depletion of nutrients (or part of the nutrients) in themedium is compensated by controlled feeding of concentrated nutrients.

It can be advantageous to adjust the pH value of the medium duringmultiplication of cells before infection to a value between pH 6.6 andpH 7.8 and especially between a value between pH 7.2 and pH 7.3.Culturing of cells preferably occurs at a temperature between 30 and 40°C. When culturing the infected cells (step b), the cells are preferablycultured at a temperature of between 30° C. and 36° C. or between 32° C.and 34° C. or at 33° C. This is particularly preferred, as it has beenshown that incubation of infected cells in this temperature rangeresults in production of a virus that results in improved efficacy whenformulated into a vaccine [35].

Oxygen partial pressure can be adjusted during culturing beforeinfection preferably at a value between 25% and 95% and especially at avalue between 35% and 60%. The values for the oxygen partial pressurestated in the context of the invention are based on saturation of air.Infection of cells occurs at a cell density of preferably about 8-25×10⁵cells/mL in the batch system or preferably about 5-20×10⁶ cells/mL inthe perfusion system. The cells can be infected with a viral dose (MOIvalue, “multiplicity of infection”; corresponds to the number of virusunits per cell at the time of infection) between 10⁻⁸ and 10, preferablybetween 0.0001 and 0.5.

Virus may be grown on cells in adherent culture or in suspension.Microcarrier cultures can be used. In some embodiments, the cells may beadapted for growth in suspension.

The methods according to the invention also include harvesting andisolation of viruses or the proteins generated by them. During isolationof viruses or proteins, the cells are separated from the culture mediumby standard methods like separation, filtration or ultrafiltration. Theviruses or the proteins are then concentrated according to methodssufficiently known to those skilled in the art, like gradientcentrifugation, filtration, precipitation, chromatography, etc., andthen purified. It is also preferred according to the invention that theviruses are inactivated during or after purification. Virus inactivationcan occur, for example, by β-propiolactone or formaldehyde at any pointwithin the purification process.

Vaccine

Vaccines (particularly for influenza virus) are generally based eitheron live virus or on inactivated virus. Inactivated vaccines may be basedon whole virions, ‘split’ virions, or on purified surface antigens.Antigens can also be presented in the form of virosomes. The inventioncan be used for manufacturing any of these types of vaccine butinactivated vaccines are preferred.

Where an inactivated virus is used, the vaccine may comprise wholevirion, split virion, or purified surface antigens (for influenza,including hemagglutinin and, usually, also including neuraminidase).Chemical means for inactivating a virus include treatment with aneffective amount of one or more of the following agents: detergents,formaldehyde, β-propiolactone, methylene blue, psoralen,carboxyfullerene (C60), binary ethylamine, acetyl ethyleneimine, orcombinations thereof. Non-chemical methods of viral inactivation areknown in the art, such as for example UV light or gamma irradiation.

Virions can be harvested from virus-containing fluids, e.g. allantoicfluid or cell culture supernatant, by various methods. For example, apurification process may involve zonal centrifugation using a linearsucrose gradient solution that includes detergent to disrupt thevirions. Antigens may then be purified, after optional dilution, bydiafiltration.

Split virions are obtained by treating purified virions with detergents(e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate,Triton X-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9,etc.) to produce subvirion preparations, including the ‘Tween-ether’splitting process. Methods of splitting influenza viruses, for exampleare well known in the art e.g. see refs. 36-41, etc. Splitting of thevirus is typically carried out by disrupting or fragmenting whole virus,whether infectious or non-infectious with a disrupting concentration ofa splitting agent. The disruption results in a full or partialsolubilisation of the virus proteins, altering the integrity of thevirus. Preferred splitting agents are non-ionic and ionic (e.g.cationic) surfactants e.g. alkylglycosides, alkylthioglycosides, acylsugars, sulphobetaines, betains, polyoxyethylenealkylethers,N,N-dialkyl-Glucamides, Hecameg, alkylphenoxy-polyethoxyethanols, NP9,quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammoniumbromides), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammoniumsalts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxypolyoxyethanols (e.g. the Triton surfactants, such as Triton X-100 orTriton N101), polyoxyethylene sorbitan esters (the Tween surfactants),polyoxyethylene ethers, polyoxyethlene esters, etc. One useful splittingprocedure uses the consecutive effects of sodium deoxycholate andformaldehyde, and splitting can take place during initial virionpurification (e.g. in a sucrose density gradient solution). Thus asplitting process can involve clarification of the virion-containingmaterial (to remove non-virion material), concentration of the harvestedvirions (e.g. using an adsorption method, such as CaHPO₄ adsorption),separation of whole virions from non-virion material, splitting ofvirions using a splitting agent in a density gradient centrifugationstep (e.g. using a sucrose gradient that contains a splitting agent suchas sodium deoxycholate), and then filtration (e.g. ultrafiltration) toremove undesired materials. Split virions can usefully be resuspended insodium phosphate-buffered isotonic sodium chloride solution. Examples ofsplit influenza vaccines are the BEGRIVAC™ FLUARIX™, FLUZONE™ andFLUSHIELD™ products.

Purified influenza virus surface antigen vaccines comprise the surfaceantigens hemagglutinin and, typically, also neuraminidase. Processes forpreparing these proteins in purified form are well known in the art. TheFLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are influenza subunitvaccines.

Another form of inactivated antigen is the virosome [42] (nucleic acidfree viral-like liposomal particles). Virosomes can be prepared bysolubilization of virus with a detergent followed by removal of thenucleocapsid and reconstitution of the membrane containing the viralglycoproteins. An alternative method for preparing virosomes involvesadding viral membrane glycoproteins to excess amounts of phospholipids,to give liposomes with viral proteins in their membrane.

The methods of the invention may also be used to produce live vaccines.Such vaccines are usually prepared by purifying virions fromvirion-containing fluids. For example, the fluids may be clarified bycentrifugation, and stabilized with buffer (e.g. containing sucrose,potassium phosphate, and monosodium glutamate). Various forms ofinfluenza virus vaccine are currently available (e.g. see chapters 17 &18 of reference 43). Live virus vaccines include MedImmune's FLUMIST™product.

The virus may be attenuated. The virus may be temperature-sensitive. Thevirus may be cold-adapted. These three features are particularly usefulwhen using live virus as an antigen.

HA is the main immunogen in current inactivated influenza vaccines, andvaccine doses are standardised by reference to HA levels, typicallymeasured by SRID. Existing vaccines typically contain about 15 μg of HAper strain, although lower doses can be used e.g. for children, or inpandemic situations, or when using an adjuvant. Fractional doses such as½ (i.e. 7.5 μg HA per strain), ¼ and ⅛ have been used, as have higherdoses (e.g. 3× or 9× doses [44,45]). Thus vaccines may include between0.1 and 150 μg of HA per influenza strain, preferably between 0.1 and 50μg e.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc.Particular doses include e.g. about 45, about 30, about 15, about 10,about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc.per strain.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content, and a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10⁶⁵-10⁷⁵) per strain is typical.

Influenza strains used with the invention may have a natural HA as foundin a wild-type virus, or a modified HA. For instance, it is known tomodify HA to remove determinants (e.g. hyper-basic regions around theHA1/HA2 cleavage site) that cause a virus to be highly pathogenic inavian species. The use of reverse genetics facilitates suchmodifications.

As well as being suitable for immunizing against inter-pandemic strains,the vaccines of the invention are particularly useful for immunizingagainst pandemic or potentially-pandemic strains. The invention issuitable for vaccinating humans as well as non-human animals.

Vaccines of the invention may include antigen(s) from one or more (e.g.1, 2, 3, 4 or more) influenza virus strains, including influenza A virusand/or influenza B virus provided that at least one influenza strain isa reassortant influenza strain of the invention. Vaccines wherein twoantigens are from a reassortant influenza strain of the invention arealso envisioned. Where a vaccine includes more than one strain ofinfluenza, the different strains are typically grown separately and aremixed after the viruses have been harvested and antigens have beenprepared. Thus a process of the invention may include the step of mixingantigens from more than one influenza strain. A trivalent vaccine istypical, including antigens from two influenza A virus strains and oneinfluenza B virus strain. A tetravalent vaccine is also useful [46],including antigens from two influenza A virus strains and two influenzaB virus strains (preferably two influenza B strains of differentlineages), or three influenza A virus strains and one influenza B virusstrain. Where the influenza vaccine includes antigens from more than oneinfluenza B strain one or more of these may be derived from areassortant influenza B virus of the invention.

Vaccines of the invention are pharmaceutically acceptable. They usuallyinclude components in addition to the antigens e.g. they typicallyinclude one or more pharmaceutical carrier(s) and/or excipient(s). Asdescribed below, adjuvants may also be included. A thorough discussionof such components is available in reference 47.

Vaccines will generally be in aqueous form. However, some vaccines maybe in dry form, e.g. in the form of injectable solids or dried orpolymerized preparations on a patch.

Vaccines may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e. less than 5 μg/ml) mercurial material e.g.thiomersal-free [40,48]. Vaccines containing no mercury are morepreferred. An α-tocopherol succinate can be included as an alternativeto mercurial compounds [40]. Preservative-free vaccines are particularlypreferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Vaccines will generally have an osmolality of between 200 mOsm/kg and400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [49], but keeping osmolality in this range is neverthelesspreferred.

Vaccines may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer (particularly with an aluminum hydroxide adjuvant); ora citrate buffer. Buffers will typically be included in the 5-20 mMrange.

The pH of a vaccine will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.A process of the invention may therefore include a step of adjusting thepH of the bulk vaccine prior to packaging.

The vaccine is preferably sterile. The vaccine is preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose. The vaccine is preferablygluten-free.

Vaccines of the invention may include detergent e.g. a polyoxyethylenesorbitan ester surfactant (known as ‘Tweens’), an octoxynol (such asoctoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), a cetyltrimethyl ammonium bromide (‘CTAB’), or sodium deoxycholate,particularly for a split or surface antigen vaccine. The detergent maybe present only at trace amounts. Thus the vaccine may include less than1 mg/ml of each of octoxynol-10 and polysorbate 80. Other residualcomponents in trace amounts could be antibiotics (e.g. neomycin,kanamycin, polymyxin B).

A vaccine may include material for a single immunisation, or may includematerial for multiple immunisations (i.e. a ‘multidose’ kit). Theinclusion of a preservative is preferred in multidose arrangements. Asan alternative (or in addition) to including a preservative in multidosevaccines, the vaccines may be contained in a container having an asepticadaptor for removal of material.

Influenza vaccines are typically administered in a dosage volume ofabout 0.5 ml, although a half dose (i.e. about 0.25 ml) may beadministered to children.

Vaccines and kits are preferably stored at between 2° C. and 8° C. Theyshould not be frozen. They should ideally be kept out of direct light.

Host Cell DNA

Where virus has been isolated and/or grown on a cell line, it isstandard practice to minimize the amount of residual cell line DNA inthe final vaccine, in order to minimize any potential oncogenic activityof the DNA.

Thus a vaccine of the invention preferably contains less than 10 ng(preferably less than 1 ng, and more preferably less than 100 pg) ofresidual host cell DNA per dose, although trace amounts of host cell DNAmay be present.

It is preferred that the average length of any residual host cell DNA isless than 500 bp e.g. less than 400 bp, less than 300 bp, less than 200bp, less than 100 bp, etc.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination is disclosed in references 50 & 51, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Treatment with an alkylatingagent, such as β-propiolactone, can also be used to remove host cellDNA, and advantageously may also be used to inactivate virions [52].

Adjuvants

Vaccines of the invention may advantageously include an adjuvant, whichcan function to enhance the immune responses (humoral and/or cellular)elicited in a subject who receives the vaccine. Preferred adjuvantscomprise oil-in-water emulsions. Various such adjuvants are known, andthey typically include at least one oil and at least one surfactant,with the oil(s) and surfactant(s) being biodegradable (metabolisable)and biocompatible. The oil droplets in the emulsion are generally lessthan 5 μm in diameter, and ideally have a sub-micron diameter, withthese small sizes being achieved with a microfluidiser to provide stableemulsions. Droplets with a size less than 220 nm are preferred as theycan be subjected to filter sterilization.

The emulsion can comprise oils such as those from an animal (such asfish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g. obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoids known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also a preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Another preferred oil isα-tocopherol (see below).

Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are Tween 80(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate),lecithin and Triton X-100.

Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Where the vaccine contains a split virus, it is preferred that itcontains free surfactant in the aqueous phase. This is advantageous asthe free surfactant can exert a ‘splitting effect’ on the antigen,thereby disrupting any unsplit virions and/or virion aggregates thatmight otherwise be present. This can improve the safety of split virusvaccines [53].

Preferred emulsions have an average droplets size of <1 μm e.g. ≤750 nm,≤500 nm, ≤400 nm, ≤300 nm, ≤250 nm, ≤220 nm, ≤200 nm, or smaller. Thesedroplet sizes can conveniently be achieved by techniques such asmicrofluidisation.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% Span 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% Span 85. This adjuvant is known as ‘MF59’ [54-56], as        described in more detail in Chapter 10 of ref. 57 and chapter 12        of ref. 58. The MF59 emulsion advantageously includes citrate        ions e.g. 10 mM sodium citrate buffer.    -   An emulsion comprising squalene, a tocopherol, and        polysorbate 80. The emulsion may include phosphate buffered        saline. These emulsions may have by volume from 2 to 10%        squalene, from 2 to 10% tocopherol and from 0.3 to 3%        polysorbate 80, and the weight ratio of squalene:tocopherol is        preferably <1 (e.g. 0.90) as this can provide a more stable        emulsion.

Squalene and polysorbate 80 may be present in a volume ratio of about5:2 or at a weight ratio of about 11:5. Thus the three components(squalene, tocopherol, polysorbate 80) may be present at a weight ratioof 1068:1186:485 or around 55:61:25. One such emulsion (‘AS03’) can bemade by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5 g of DL a tocopherol and 5 mlsqualene), then microfluidising the mixture. The resulting emulsion mayhave submicron oil droplets e.g. with an average diameter of between 100and 250 nm, preferably about 180 nm. The emulsion may also include a3-de-O-acylated monophosphoryl lipid A (3d MPL). Another useful emulsionof this type may comprise, per human dose, 0.5-10 mg squalene, 0.5-11 mgtocopherol, and 0.1-4 mg polysorbate 80 [59] e.g. in the ratiosdiscussed above.

-   -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see        below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“Pluronic™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant [60] (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant [61]        (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).        Microfluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [62]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. The emulsion may        include a TLR4 agonist [63]. Such emulsions may be lyophilized    -   An emulsion of squalene, poloxamer 105 and Abil-Care [64]. The        final concentration (weight) of these components in adjuvanted        vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and        2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone;        caprylic/capric triglyceride).    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 65, preferred phospholipid components are        phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   A submicron oil-in-water emulsion of a non-metabolisable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, Tween 80 or Span 80). Additives may be included, such        as QuilA saponin, cholesterol, a saponin-lipophile conjugate        (such as GPI-0100, described in reference 66, produced by        addition of aliphatic amine to desacylsaponin via the carboxyl        group of glucuronic acid), dimethyidioctadecylammonium bromide        and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [67].    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [68].    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [68].

In some embodiments an emulsion may be mixed with antigenextemporaneously, at the time of delivery, and thus the adjuvant andantigen may be kept separately in a packaged or distributed vaccine,ready for final formulation at the time of use. In other embodiments anemulsion is mixed with antigen during manufacture, and thus thecomposition is packaged in a liquid adjuvanted form. The antigen willgenerally be in an aqueous form, such that the vaccine is finallyprepared by mixing two liquids.

The volume ratio of the two liquids for mixing can vary (e.g. between5:1 and 1:5) but is generally about 1:1. Where concentrations ofcomponents are given in the above descriptions of specific emulsions,these concentrations are typically for an undiluted composition, and theconcentration after mixing with an antigen solution will thus decrease.

Packaging of Vaccines

Suitable containers for vaccines of the invention (or kit components)include vials, syringes (e.g. disposable syringes), nasal sprays, etc.These containers should be sterile.

Where a composition/component is located in a vial, the vial ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials are preferably sealed with a latex-freestopper, and the absence of latex in all packaging material ispreferred. The vial may include a single dose of vaccine, or it mayinclude more than one dose (a ‘multidose’ vial) e.g. 10 doses. Preferredvials are made of colourless glass. A vial may have a cap that permitsaseptic removal of its contents, particularly for multidose vials.

Where a component is packaged into a syringe, the syringe may have aneedle attached to it. If a needle is not attached, a separate needlemay be supplied with the syringe for assembly and use. Such a needle maybe sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch25-gauge and ⅝-inch 25-gauge needles are typical. Syringes may beprovided with peel-off labels on which the lot number, influenza seasonand expiration date of the contents may be printed, to facilitate recordkeeping. The plunger in the syringe preferably has a stopper to preventthe plunger from being accidentally removed during aspiration. Thesyringes may have a latex rubber cap and/or plunger. Disposable syringescontain a single dose of vaccine. The syringe will generally have a tipcap to seal the tip prior to attachment of a needle, and the tip cap ispreferably made of a butyl rubber. If the syringe and needle arepackaged separately then the needle is preferably fitted with a butylrubber shield. Preferred syringes are those marketed under the tradename “Tip-Lok”™.

Containers may be marked to show a half-dose volume e.g. to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

A kit or vaccine may be packaged (e.g. in the same box) with a leafletincluding details of the vaccine e.g. instructions for administration,details of the antigens within the vaccine, etc. The instructions mayalso contain warnings e.g. to keep a solution of adrenaline readilyavailable in case of anaphylactic reaction following vaccination, etc.

Methods of Treatment, and Administration of the Vaccine

The invention provides a vaccine manufactured according to theinvention. These vaccines are suitable for administration to human ornon-human animal subjects, such as pigs or birds, and the inventionprovides a method of raising an immune response in a subject, comprisingthe step of administering a vaccine of the invention to the subject. Theinvention also provides a vaccine of the invention for use as amedicament, and provides the use of a vaccine of the invention for themanufacture of a medicament for raising an immune response in a subject.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses, neutralising capability andprotection after influenza virus vaccination are well known in the art.Human studies have shown that antibody titers against hemagglutinin ofhuman influenza virus are correlated with protection (a serum samplehemagglutination-inhibition titer of about 30-40 gives around 50%protection from infection by a homologous virus) [69]. Antibodyresponses are typically measured by hemagglutination inhibition, bymicroneutralisation, by single radial immunodiffusion (SRID), and/or bysingle radial hemolysis (SRH). These assay techniques are well known inthe art.

Vaccines of the invention can be administered in various ways. The mostpreferred immunisation route is by intramuscular injection (e.g. intothe arm or leg), but other available routes include subcutaneousinjection, intranasal [70-72], oral [73], intradermal [74,75],transcutaneous, transdermal [76], etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Influenza vaccines are currently recommended foruse in pediatric and adult immunisation, from the age of 6 months. Thusa human subject may be less than 1 year old, 1-5 years old, 5-15 yearsold, 15-55 years old, or at least 55 years old. Preferred subjects forreceiving the vaccines are the elderly (e.g. ≥50 years old, ≥60 yearsold, and preferably ≥65 years), the young (e.g. ≤5 years old),hospitalised subjects, healthcare workers, armed service and militarypersonnel, pregnant women, the chronically ill, immunodeficientsubjects, subjects who have taken an antiviral compound (e.g. anoseltamivir or zanamivir compound; see below) in the 7 days prior toreceiving the vaccine, people with egg allergies and people travellingabroad. The vaccines are not suitable solely for these groups, however,and may be used more generally in a population. For pandemic strains,administration to all age groups is preferred.

Preferred vaccines of the invention satisfy 1, 2 or 3 of the CPMPcriteria for efficacy. In adults (18-60 years), these criteria are: (1)≥70% seroprotection; (2) ≥40% seroconversion; and/or (3) a GMT increaseof ≥2.5-fold. In elderly (>60 years), these criteria are: (1) ≥60%seroprotection; (2) ≥30% seroconversion; and/or (3) a GMT increase of≥2-fold. These criteria are based on open label studies with at least 50patients.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naïve patients e.g. for peoplewho have never received an influenza vaccine before, or for vaccinatingagainst a new HA subtype (as in a pandemic outbreak). Multiple doseswill typically be administered at least 1 week apart (e.g. about 2weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about10 weeks, about 12 weeks, about 16 weeks, etc.).

Vaccines produced according to the invention may be administered topatients at substantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines e.g. at substantially the same time as a measlesvaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicellavaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a DTP vaccine, a conjugated H. influenzae type bvaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine,a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Yvaccine), a respiratory syncytial virus vaccine, a pneumococcalconjugate vaccine, etc. Administration at substantially the same time asa pneumococcal vaccine and/or a meningococcal vaccine is particularlyuseful in elderly patients.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g. oseltamivir and/or zanamivir). These antiviralsinclude neuraminidase inhibitors, such as a(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate(TAMIFLU™).

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

The various steps of the methods may be carried out at the same ordifferent times, in the same or different geographical locations, e.g.countries, and by the same or different people or entities.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 ofreference 77. A preferred alignment is determined by the Smith-Watermanhomology search algorithm using an affine gap search with a gap openpenalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is taught in reference 78.

References to a percentage sequence identity between two nucleic acidsequences mean that, when aligned, that percentage of bases are the samein comparing the two sequences. This alignment and the percent homologyor sequence identity can be determined using software programs known inthe art, for example those described in section 7.7.18 of reference 77.A preferred alignment program is GCG Gap (Genetics Computer Group,Wisconsin, Suite Version 10.1), preferably using default parameters,which are as follows: open gap=3; extend gap=1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the HA yield of different reassortant influenza Bstrains in MDCK cells relative to the wild-type (WT) or reversegenetics-derived (RG) B/Brisbane/60/08 strain. The viral segments of thetested influenza B viruses are shown in Table 1. The y-axis indicatesthe HA yield in μg/mL.

FIG. 2 compares the HA yield of different reassortant influenza Bstrains in MDCK cells relative to the wild-type (WT) or reversegenetics-derived (RG) B/Panama/45/90 strain. The viral segments of thetested influenza B viruses are shown in Table 1. The y-axis indicatesthe HA yield in μg/mL.

FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D compare the HA yield ofreassortant influenza B viruses comprising the B/Panama/45/90 orB/Brisbane/60/08 backbone with the HA yield obtained with thecorresponding wild-type strain. The different experiments were performedusing the B/Brisbane/60/08 HA and NA segments FIG. 3A, theB/Panama/45/90 HA and NA segments FIG. 3B, the B/Florida/4/06 HA and NAsegments FIG. 3C or the B/Lee/40 HA and NA segments FIG. 3D. The whitebar shows the results with wild-type-strain, the cross-hatched barindicates the results with the B/Panama/45/90 backbone and the checkedbar shows the results with the B/Brisbane/60/08 backbone. The y-axis inFIG. 3A, FIG. 3B, and FIG. 3C indicates the HA yield in μg/mL asdetermined by ELISA and the y-axis in FIG. 3D shows the HA titre asdetermined by HA assay.

FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D compare the HA yield ofreassortant influenza B viruses comprising the #2, #9, #32, or #34hybrid backbone (as shown in Table 1) with the HA yield obtained withthe BX-35, B/Panama/45/90, or B/Brisbane/60/08 backbone or thecorresponding wild-type virus. The different experiments were performedusing the B/Brisbane/60/08 HA and NA segments FIG. 4A, theB/Panama/45/90 HA and NA segments FIG. 4B, the BX-35 HA and NA segmentsFIG. 4C or the B/Florida/4/06 HA and NA segments FIG. 4D. The y-axisindicates the HA yield in μg/mL.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, and FIG.5H compare the HA yield of reassortant influenza B viruses comprisingthe #34 or B/Brisbane/60/08 backbones with the HA yield obtained withthe corresponding wild-type strain. The different experiments wereperformed using the B/Panama/45/90 HA and NA segments FIG. 5A, theB/Brisbane/60/08 HA and NA segments FIG. 5B, the B/Florida/4/06 HA andNA segments FIG. 5C, the B/Brisbane/03/07 HA and NA segments FIG. 5D,the B/Brisbane/32/02 HA and NA segments FIG. 5E, the BX-35 HA and NAsegments FIG. 5F, the B/Malaysia/2506/04 HA and NA segments FIG. 5G, orthe B/Hubei-Wujiagang/159/08 HA and NA segments FIG. 5H. The white barshows the results with the B/Brisbane/60/08 backbone, the cross-hatchedbar indicates the results with the #34 backbone and the checked barshows the results with the wild-type strain. The y-axis indicates the HAyield in μg/mL.

FIG. 6A and FIG. 6B compare the HA titre FIG. 6A and the viral growthFIG. 6B of (a) a reverse genetics-derived reassortant influenza B viruscomprising all backbone segments from B/Brisbane/60/08 (#35) and the HAand NA segments from B/Wisconsin/1/10, (b) a reassortant influenza Bvirus which comprises the PB2, NP and M segments from B/Lee/40 and allother genes from B/Wisconsin/1/10 (#41), and (c) the wild-typeB/Wisconsin/1/10 strain (WT) following growth in embryonated chickeneggs. Each triangle represents an individual egg and the bars representmean values. “280” and “2800” indicate the infectious doses (IU) withwhich the eggs were inoculated. The y-axis in FIG. 6A represents the HAyield and the y-axis in FIG. 6B represents IU/mL.

MODES FOR CARRYING OUT THE INVENTION

Development of New Donor Strains

In order to provide high-growth donor strains, the inventors found thatreassortant influenza B viruses comprising backbone segments fromB/Brisbane/60/08 and B/Panama/45/90 grow particularly well in eggs andin cells, such as MDCK cells. To this end, reassortant influenza Bviruses comprising backbone segments from these viruses are produced andthe resulting influenza B viruses are grown in MDCK cells. The viralyield is determined by ELISA (as described in PCT/IB2012/057235) or a HAassay as known in the art.

Growth Characteristics of Reassortant Influenza B Viruses

Reassortant influenza B viruses are produced by reverse genetics whichcontain the HA and NA proteins from various influenza strains and theother viral segments from B/Brisbane/60/08 and/or B/Panama/45/90. As acontrol the corresponding wild-type influenza B strain is used. Theseviruses are cultured either in embyronated chicken eggs or in MDCKcells. The following influenza B strains are used:

TABLE 1 Antigenic Backbone segments determinants combo # PA PB1 PB2 NPNS M HA NA 1 (WT) Brisbane Brisbane Brisbane Brisbane Brisbane BrisbaneBrisbane Brisbane  2 Panama Brisbane Brisbane Brisbane Brisbane BrisbaneBrisbane Brisbane  3 Brisbane Panama Brisbane Brisbane Brisbane BrisbaneBrisbane Brisbane  4 Brisbane Brisbane Panama Brisbane Brisbane BrisbaneBrisbane Brisbane  5 Brisbane Brisbane Brisbane Panama Brisbane BrisbaneBrisbane Brisbane  6 Panama Panama Brisbane Brisbane Brisbane BrisbaneBrisbane Brisbane  7 Panama Brisbane Panama Brisbane Brisbane BrisbaneBrisbane Brisbane  8 Panama Brisbane Brisbane Panama Brisbane BrisbaneBrisbane Brisbane  9 Brisbane Panama Panama Brisbane Brisbane BrisbaneBrisbane Brisbane 10 Brisbane Panama Brisbane Panama Brisbane BrisbaneBrisbane Brisbane 11 Brisbane Brisbane Panama Panama Brisbane BrisbaneBrisbane Brisbane 12 Panama Panama Panama Brisbane Brisbane BrisbaneBrisbane Brisbane 13 Panama Panama Brisbane Panama Brisbane BrisbaneBrisbane Brisbane 14 Panama Brisbane Panama Panama Brisbane BrisbaneBrisbane Brisbane 15 Brisbane Panama Panama Panama Brisbane BrisbaneBrisbane Brisbane 16 Panama Panama Panama Panama Brisbane BrisbaneBrisbane Brisbane 17 Panama Panama Panama Panama Panama Panama BrisbaneBrisbane 20 Brisbane Panama Panama Panama Panama Panama Panama Panama 21Panama Brisbane Panama Panama Panama Panama Panama Panama 22 PanamaPanama Brisbane Panama Panama Panama Panama Panama 23 Panama PanamaPanama Brisbane Panama Panama Panama Panama 24 Brisbane Brisbane PanamaPanama Panama Panama Panama Panama 25 Brisbane Panama Brisbane PanamaPanama Panama Panama Panama 26 Brisbane Panama Panama Brisbane PanamaPanama Panama Panama 27 Panama Brisbane Brisbane Panama Panama PanamaPanama Panama 28 Panama Brisbane Panama Brisbane Panama Panama PanamaPanama 29 Panama Panama Brisbane Brisbane Panama Panama Panama Panama 30Brisbane Brisbane Brisbane Panama Panama Panama Panama Panama 31Brisbane Brisbane Panama Brisbane Panama Panama Panama Panama 32Brisbane Panama Brisbane Brisbane Panama Panama Panama Panama 33 PanamaBrisbane Brisbane Brisbane Panama Panama Panama Panama 34 BrisbaneBrisbane Brisbane Brisbane Panama Panama Panama Panama 35 BrisbaneBrisbane Brisbane Brisbane Brisbane Brisbane Panama Panama

The results indicate that reassortant viruses #2, #9, #22, #23, #29,#30, #31, #32, #33, #34 and #35 grow equally well or even better in theculture host (see FIGS. 1 and 2) than the corresponding wild-typestrain. Most of these strains comprise the NP segment fromB/Brisbane/60/08 and some (in particular those which grew best) furthercontain the PB2 segment from B/Brisbane/60/08. All of these viruses alsocontain viral segments from the B/Victoria/2/87-like strain and theB/Yamagata/16/88-like strain at a ratio 7:1, 6:2, 4:4, 3:4 or 1:7.

Growth Characteristics of Reassortant Influenza B Viruses ComprisingBackbone Segments From B/Brisbane/60/08

In order to test whether the reassortant influenza B viruses of theinvention can be used with HA and NA segments from different strains,reassortant influenza B viruses comprising the HA and NA segments fromB/Panama/45/90, B/Lee/40 or B/Florida/04/06 and the backbone segmentsfrom B/Brisbane/60/08 are produced. The reassortant influenza virusesare grown in MDCK cells for 60 hours and the HA yield is determined byELISA or a HA assay. The data (see FIG. 3A, FIG. 3B, FIG. 3C, and FIG.3D) show that all of the reassortant influenza B viruses grew to highertitres compared to the wild-type influenza B viruses which indicatesthat reassortant influenza B viruses of the invention are useful for avariety of different HA and NA segments.

Growth Characteristics of Reassortant Influenza B Viruses ComprisingHybrid Backbone Segments

The growth characteristics of reassortant influenza B viruses comprisingthe backbone segments of the invention are also determined relative tothe wild-type influenza B virus and the known influenza B reassortantBX35 which comprises the HA, NA, PA, PBI, and NS segments fromB/Brisbane/60/08, the PB2 and M segments from B/Panama/45/90, and the NPsegment from B/Lee/40. The backbones are tested with the HA and NAproteins of B/Brisbane/60/08, B/Panama/45/90, BX-35 and B/Florida/04/06.The reassortant influenza viruses are grown in MDCK cells for 60 hoursand the HA yield is determined by ELISA or RP-HPLC. The data (see FIG.4A, FIG. 4B, FIG. 4C, and FIG. 4D) show that reassortant influenza Bviruses comprising the backbones of the invention can grow to highertitres compared to the wild-type and to reassortant influenza B virusescomprising the known BX35 backbone.

The HA yield obtained with the #34 and #35 is further tested using anumber of different HA and NA segments. MDCK cells are infected withthese reassortant influenza B viruses and the corresponding wild-typeinfluenza B virus. The data (see FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D,FIG. 5E, FIG. 5F, FIG. 5G, and FIG. 5H) show that all of the reassortantinfluenza B viruses comprising a backbone according to the invention cangrow equally well or better than the corresponding wild-type strain.

Growth Characteristics of Reassortant Influenza B Viruses in Eggs

Embryonated chicken eggs were inoculated with either 280 or 2800infectious doses (IU) of (a) a reverse genetics-derived reassortantinfluenza B virus comprising all backbone segments from B/Brisbane/60/08(#35 backbone) and the HA and NA segments from B/Wisconsin/1/10, (b) areassortant influenza B virus which comprises PB2, NP and M segmentsfrom B/Lee/40 and all other genes from B/Wisconsin/1/10 (BX-41), and (c)the wild-type B/Wisconsin/1/10 strain. Egg allantoic fluid was harvested72h post-infection, and assayed for HA titer by HA Assay or virus growthby a focus-formation assay. The data (see FIG. 6A and FIG. 6B) show thatthe reassortant influenza B virus according to the invention can growequally well or better than the control strains.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

-   [1] WO2007/002008-   [2] WO2007/124327-   [3] Goodeve et al. (1985) Arch Virol. 83(3-4):169-79.-   [4] Audsley, J 2007, Alternative approaches in the preparation and    growth of influenza B vaccine-   viruses, PhD Thesis, School of Applied Sciences, RMIT University.-   [5]    http://www.who.int/influenza/vaccines/virus/candidates_reagents/summary_b_cvv_20120621.pdf-   [6] http://www.nibsc.ac.uk/documents/ifu/09-318.pdf-   [7] http://www.nibsc.ac.uk/documents/ifu/12-202.pdf-   [8] Rota et al. (1992) J Gen Virol 73:2737-42.-   [9] GenBank sequence GI:325176.-   [10] McCullers et al. (1999) J Virol 73:7343-8.-   [11] GenBank sequence GI:325237.-   [12] Herlocher et al. (2004) J Infect Dis 190(9):1627-30.-   [13] Le et al. (2005) Nature 437(7062):1108.-   [14] U.S. Pat. No. 6,468,544-   [15] Neumann et al. (2005) Proc Natl Acad Sci USA 102: 16825-9-   [16] WO2010/133964-   [17] WO2009/000891-   [18] U.S. provisional application No. 61/273,151-   [19] Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 ed.,    1989, Cold Spring Harbor Press, Cold Spring Harbor, N. Y-   [20] WO2011/012999-   [21] WO2011048560-   [22] Kistner et al. (1998) Vaccine 16:960-8.-   [23] Kistner et al. (1999) Dev Biol Stand 98:101-110.-   [24] Bruhl et al. (2000) Vaccine 19:1149-58.-   [25] WO2006/108846.-   [26] Pau et al. (2001) Vaccine 19:2716-21.-   [27] http://www.atcc.org/[28]-   [28] http://locus.umdnj.edu/[29]-   [29] WO97/37000.-   [30] Brands et al. (1999) Dev Biol Stand 98:93-100.-   [31] Halperin et al. (2002) Vaccine 20:1240-7.-   [32] EP-A-1260581 (WO01/64846)-   [33] WO2006/071563-   [34] WO2005/113758-   [35] WO97/37001-   [36] WO02/28422.-   [37] WO02/067983.-   [38] WO02/074336.-   [39] WO01/21151.-   [40] WO02/097072.-   [41] WO2005/113756.-   [42] Huckriede et al. (2003) Methods Enzymol 373:74-91.-   [43] Vaccines. (eds. Plotkins & Orenstein). 4th edition, 2004, ISBN:    0-7216-9688-0-   [44] Treanor et al. (1996) J Infect Dis 173:1467-70.-   [45] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10.-   [46] WO2008/068631.-   [47] Gennaro (2000) Remington: The Science and Practice of Pharmacy.    20th edition, ISBN: 0683306472.-   [48] Banzhoff (2000) Immunology Letters 71:91-96.-   [49] Nony et al. (2001) Vaccine 27:3645-51.-   [50] EP-B-0870508.-   [51] U.S. Pat. No. 5,948,410.-   [52] WO2007/052163.-   [53] WO2007/052061-   [54] WO90/14837.-   [55] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203.-   [56] Podda (2001) Vaccine 19: 2673-2680.-   [57] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell    & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).-   [58] Vaccine Adjuvants: Preparation Methods and Research Protocols    (Volume 42 of Methods in Molecular Medicine series). ISBN:    1-59259-083-7. Ed. O'Hagan.-   [59] WO2008/043774.-   [60] Allison & Byars (1992) Res Immunol 143:519-25.-   [61] Hariharan et al. (1995) Cancer Res 55:3486-9.-   [62] U.S.-2007/014805.-   [63] U.S.-2007/0191314.-   [64] Suli et al. (2004) Vaccine 22(25-26):3464-9.-   [65] WO95/11700.-   [66] U.S. Pat. No. 6,080,725.-   [67] WO2005/097181.-   [68] WO2006/113373.-   [69] Potter & Oxford (1979) Br Med Bull 35: 69-75.-   [70] Greenbaum et al. (2004) Vaccine 22:2566-77.-   [71] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304.-   [72] Piascik (2003) J Am Pharm Assoc (Wash D.C.). 43:728-30.-   [73] Mann et al. (2004) Vaccine 22:2425-9.-   [74] Halperin et al. (1979) Am J Public Health 69:1247-50.-   [75] Herbert et al. (1979) J Infect Dis 140:234-8.-   [76] Chen et al. (2003) Vaccine 21:2830-6.-   [77] Current Protocols in Molecular Biology (F. M. Ausubel et al.,    eds., 1987) Supplement 30.-   [78] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.

SEQUENCES (PA, B/Brisbane/60/08) SEQ ID NO: 1MDTFITRNFQTTIIQKAKNTMAEFSEDPELQPAMLFNICVHLEVCYVISDMNFLDEEGKAYTALEGQGKEQNLRPQYEVIEGMPRTIAWMVQRSLAQEHGIETPKYLADLFDYKTKRFIEVGITKGLADDYFWKKKEKLGNSMELMIFSYNQDYSLSNESSLDEEGKGRVLSRLTELQAELSLKNLWQVLIGEEDVEKGIDFKLGQTISRLRDISVPAGFSNFEGMRSYIDNIDPKGAIERNLARMSPLVSVTPKKLTWEDLRPIGPHIYDHELPEVPYNAFLLMSDELGLANMTEGKSKKPKTLAKECLEKYSTLRDQTDPILIMKSEKANENFLWKLWRDCVNTISNEETSNELQKTNYAKWATGDGLTYQKIMKEVAIDDETMCQEEPKIPNKCRVAAWVQTEMNLLSTLTSKRALDLPEIGPDIAPVEHVGSERRKYFVNEINYCKASTVMMKYVLFHTSLLNESNASMGKYKVIPITNRVVNEKGESFDMLYGLAVKGQSHLRGDTDVVTVVTFEFSSTDPRVDSGKWPKYTVFRIGSLFVSGREKSVYLYCRVNGTNKIQMKWGMEARRCLLQSMQQMEAIVEQESSIQGYDMTKACFKGDRVNSPKTFSIGTQEGKLVKGSFGKALRVIFTKCLMHYVFGNAQLEGFSAESRRLLLLIQALKDRKGPWVFDLEGMYSGIEECISNNPWVIQSVYWFNEWLGFEKEGNKVLESVDEIMDE (PB1, B/Brisbane/60/08) SEQ ID NO: 2MNINPYFLFIDVPVQAAISTTFPYTGVPPYSHGTGTGYTIDTVIRTHEYSNKGKQYISDVTGCTMVDPTNGPLPEDNEPSAYAQLDCVLEALDRMDEEHPGLFQAASQNAMEALMVTTVDKLTQGRQTFDWTVCRNQPAATALNTTITSFRLNDLNGADKGGLIPFCQDIIDSLDRPEMTFFSVKNIKKKLPAKNRKGFLIKRIPMKVKDKITKVEYIKRALSLNTMTKDAERGKLKRRAIATAGIQIRGFVLVVENLAKNICENLEQSGLPVGGNEKKAKLSNAVAKMLSNCPPGGISMTVTGDNTKWNECLNPRIFLAMTERITRDSPVWFRDFCSIAPVLFSNKIARLGKGFMITSKTKRLKAQIPCPDLFSIPLERYNEETRAKLKKLKPFFNEEGTASLSPGMMMGMFNMLSTVLGVAALGIKNIGNKEYLWDGLQSSDDFALFVNAKDEETCMEGINDFYRTCKLLGVNMSKKKSYCNETGMFEFTSMFYRDGFVSNFAMELPSFGVAGVNESADMAIGMTIIKNNMINNGMGPATAQTAIQLFIADYRYTYKCHRGDSKVEGKRMKIIKELWENTKGRDGLLVADGGPNIYNLRNLHIPEIVLKYNLMDPEYKGRLLHPQNPFVGHLSIEGIKEADITPAHGPVKKMDYDAVSGTHSWRTKRNRSILNTDQRNMILEEQCYAKCCNLFEACFNSASYRKPVGQHSMLEAMAHRLRMDARLDYESGRMSKDDFEKAMAHLGEIGYI(PB2, B/Brisbane/60/08) SEQ ID NO: 3MTLAKIELLKQLLRDNEAKTVLKQTTVDQYNIIRKFNTSRIEKNPSLRMKWAMCSNFPLALTKGDMANRIPLEYKGIQLKTNAEDIGTKGQMCSIAAVTWWNTYGPIGDTEGFERVYESFFLRKMRLDNATWGRITFGPVERVRKRVLLNPLTKEMPPDEASNVIMEILFPKEAGIPRESTWIHRELIKEKREKLKGTMITPIVLAYMLERELVARRRFLPVAGATSAEFIEMLHCLQGENWRQIYHPGGNKLTESRSQSMIVACRKIIRRSIVASNPLELAVEIANKTVIDTEPLKSCLAAIDGGDVACDIIRAALGLKIRQRQRFGRLELKRISGRGFKNDEEILIGNGTIQKIGIWDGEEEFHVRCGECRGILKKSKMKLEKLLINSAKKEDMRDLIILCMVFSQDTRMFQGVRGEINFLNRAGQLLSPMYQLQRYFLNRSNDLFDQWGYEESPKASELHGINESMNASDYTLKGIVVTRNVIDDFSSIETEKVSITKNLSLIKRTGEVIMGANDVSELESQAQLMITYDTPKMWEMGTTKELVQNTYQWVLKNLVTLKAQFLLGKEDMFQWDAFEAFESIIPQKMAGQYSGFARAVLKQMRDQEVMKTDQFIKLLPFCFSPPKLRSNGEPYQFLKLVLKGGGENFIEVRKGSPLFSYNPQTEVLTICGRMMSLKGKIEDEERNRSMGNAVLAGFLVSGKYDPDLGDFKTIEELEKLKPGEKANILLYQGKPVKVVKRKRYSALSNDISQGIKRQRMTVESMGWALS(NP, B/Brisbane/60/08) SEQ ID NO: 4MSNMDIDGINTGTIDKTPEEITSGTSGTTRPIIRPATLAPPSNKRTRNPSPERATTSSEDDVGRKTQKKQTPTEIKKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAHAVERILLAATDDKKTEFQKKKNARDVKEGKEEIDHNKTGGTFYKMVRDDKTIYFSPIRITFLKEEVKTMYKTTMGSDGFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTVPRRSGATGVAIKGGGTLVAEAIRFIGRAMADRGLLRDIKAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRNPGIADIEDLTLLARSMVVVRPSVASKVVLPISIYAKIPQLGFNVEEYSMVGYEAMALYNMATPVSILRMGDDAKDKSQLFFMSCFGAAYEDLRVLSALTGTEFKPRSALKCKGFHVPAKEQVEGMGAALMSIKLQFWAPMTRSGGNEVGGDGGSGQISCSPVFAVERPIALSKQAVRRMLSMNIEGRDADVKGNLLKMMNDSMAKKTSGNAFIGKKMFQISDKNKTNPIEIPIKQTIPNFFFGRDTAEDYDDLDY (M₁, B/Brisbane/60/08) SEQ ID NO: 5MSLFGDTIAYLLSLTEDGEGKAELAEKLHCWFGGKEFDLDSALEWIKNKRCLTDIQKALIGASICFLKPKDQERKRRFITEPLSGMGTTATKKKGLILAERKMRRCVSFHEAFEIAEGHESSALLYCLMVMYLNPGNYSMQVKLGTLCALCEKQASHSHRAHSRAARSSVPGVRREMQMVSAMNTAKTMNGMGKGEDVQKLAEELQSNIGVLRSLGASQKNGEGIAKDVMEVLKQSSMGNSALVKKYL (M₂, B/Brisbane/60/08) SEQ ID NO: 6MLEPFQILTICSFILSALHFMAWTIGHLNQIKRGINMKIRIKGPNKETINREVSILRHSYQKEIQAKETMKEVLSDNMEVLNDHIIIEGLSAEEIIKMGETVLEIEELH (NS₁, B/Brisbane/60/08) SEQ ID NO: 7MANNNMTTTQIEVGPGATNATINFEAGILECYERLSWQRALDYPGQDRLNRLKRKLESRIKTHNKSEPESKRMSLEERKAIGVKMMKVLLFMNPSAGIEGFEPYCMKSSSNSNCTKYNWTDYPSTPERCLDDIEEEPEDVDGPTEIVLRDMNNKDARQKIKEEVNTQKEGKFRLTIKRDMRNVLSLRVLVNGTFLKHPNGHKSLSTLHRLNAYDQSGRLVAKLVATDDLTVEDEEDGHRILNSLFERLNEGHSKPIRAAETAVGVLSQFGQEHRLSPEEGDN(NS₂, B/Brisbane/60/08) SEQ ID NO: 8MANNNMTTTQIEWRMKKMAIGSSTHSSSVLMKDIQSQFEQLKLRWESYPNLVKSTDYHQKRETIRLVTEELYLLSKRIDDNILFHKTVIANSSIIADMVVSLSLLETLYEMKDVVEVYSRQCL (HA, B/Brisbane/60/08)SEQ ID NO: 9MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGEHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL (NA, B/Brisbane/60/08)SEQ ID NO: 10MLPSTIQTLTLFLTSGGVLLSLYVSASLSYLLYSDILLKFSPTEITAPTMPLDCANASNVQAVNRSATKGVTLLLPEPEWTYPRLSCPGSTFQKALLISPHRFGETKGNSAPLIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRIIKEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPCESNGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYSFGFEIKDKKCDVPCIGIEMVHDGGKETWHSAATAIYCLMGSGQLLWDTVTGVDMAL (PA, B/Brisbane/60/08) SEQ ID NO: 11AGCAGAAGCGGTGCGTTTGATTTGTCATAATGGATACTTTTATTACAAGAAACTTCCAGACTACAATAATACAAAAGGCCAAAAACACAATGGCAGAATTTAGTGAAGATCCTGAATTGCAACCAGCAATGCTATTCAATATCTGCGTCCATCTAGAGGTTTGCTATGTAATAAGTGACATGAATTTTCTTGACGAAGAAGGAAAAGCATATACAGCATTAGAAGGACAAGGGAAAGAACAAAACTTGAGACCACAATATGAAGTAATTGAGGGAATGCCAAGAACCATAGCATGGATGGTCCAGAGATCCTTAGCTCAAGAGCATGGAATAGAGACTCCCAAGTATCTGGCTGATTTGTTTGATTATAAAACCAAAAGATTTATAGAAGTTGGAATAACAAAGGGATTGGCTGATGATTACTTTTGGAAAAAGAAAGAAAAGTTGGGAAATAGCATGGAACTGATGATATTCAGCTACAATCAAGACTACTCGTTAAGTAATGAATCCTCATTGGATGAGGAAGGGAAAGGGAGAGTGCTAAGCAGACTCACAGAACTTCAGGCTGAATTAAGTCTGAAAAATTTATGGCAAGTTCTCATAGGAGAAGAAGATGTTGAAAAGGGAATTGATTTTAAACTTGGACAAACAATATCTAGACTAAGGGATATATCTGTTCCAGCTGGTTTCTCCAATTTTGAAGGAATGAGGAGCTACATAGACAATATAGACCCAAAAGGAGCAATAGAGAGAAATCTAGCAAGGATGTCTCCCTTAGTATCAGTCACACCTAAAAAGTTAACATGGGAGGACCTAAGACCAATAGGGCCTCACATTTACGACCATGAGCTACCAGAAGTTCCATATAATGCCTTTCTTCTAATGTCTGATGAACTGGGATTGGCCAATATGACTGAGGGAAAGTCCAAAAAACCGAAGACATTAGCCAAAGAATGTCTAGAAAAGTACTCAACACTACGGGATCAAACTGACCCAATATTAATAATGAAAAGCGAAAAAGCTAACGAAAATTTCCTATGGAAGCTTTGGAGAGACTGTGTAAATACAATAAGTAATGAGGAAACGAGTAACGAGTTACAGAAAACCAATTATGCCAAATGGGCCACAGGGGATGGATTAACATACCAGAAAATAATGAAAGAAGTAGCAATAGATGACGAAACAATGTGCCAAGAAGAGCCTAAAATCCCTAACAAATGTAGAGTGGCTGCTTGGGTTCAAACAGAGATGAATCTATTGAGCACTCTGACAAGTAAAAGAGCTCTGGACCTACCAGAAATAGGGCCAGACATAGCACCCGTGGAGCATGTAGGAAGTGAAAGAAGGAAATACTTTGTTAATGAAATCAACTACTGTAAGGCCTCTACAGTTATGATGAAGTATGTGCTTTTTCACACTTCATTGTTGAATGAAAGCAATGCCAGCATGGGAAAATACAAAGTAATACCAATAACCAATAGAGTAGTAAATGAAAAAGGAGAAAGTTTCGACATGCTTTACGGTCTGGCGGTTAAAGGACAATCTCATCTGAGGGGAGATACTGATGTTGTAACAGTTGTAACTTTCGAATTTAGTAGTACAGATCCAAGAGTGGACTCAGGAAAGTGGCCAAAATATACTGTGTTTAGGATTGGCTCCCTATTTGTGAGTGGGAGGGAAAAATCTGTGTACTTGTATTGCCGAGTGAATGGCACAAATAAGATCCAAATGAAATGGGGAATGGAAGCTAGAAGATGTTTGCTTCAATCAATGCAACAAATGGAGGCAATTGTTGAACAGGAATCATCAATACAAGGATATGACATGACCAAAGCCTGTTTCAAGGGAGACAGAGTAAATAGCCCCAAAACTTTCAGTATTGGAACTCAAGAAGGAAAACTAGTAAAAGGATCCTTTGGAAAAGCACTAAGAGTAATATTTACTAAATGCTTGATGCACTATGTATTTGGAAATGCCCAATTGGAGGGGTTTAGTGCCGAGTCTAGGAGACTTCTACTGTTGATTCAAGCATTAAAGGACAGAAAGGGCCCTTGGGTGTTCGACTTAGAGGGAATGTATTCTGGAATAGAAGAATGTATTAGCAACAACCCTTGGGTAATACAGAGTGTATACTGGTTCAATGAATGGTTGGGCTTTGAAAAGGAGGGGAATAAAGTGTTGGAATCAGTGGATGAAATAATGGATGAATAAAAGGAAATGGTACTCAATTTGGTACTATTTTGTTCATTATGTATCTAAACATCCAATAAAAAGAACCAAGAATCAAAAATGCACGTGTTTCTACT(PB1, B/Brisbane/60/08) SEQ ID NO: 12AGCAGAAGCGGAGCCTTTAAGATGAATATAAATCCTTATTTTCTCTTCATAGATGTGCCCGTACAGGCAGCAATTTCAACAACATTCCCATACACTGGTGTTCCCCCTTATTCCCATGGAACAGGAACAGGCTACACAATAGACACCGTGATCAGAACGCATGAGTACTCAAACAAGGGGAAACAGTACATTTCTGATGTTACAGGATGCACAATGGTAGATCCAACAAATGGACCATTACCCGAAGATAATGAGCCGAGTGCCTATGCGCAATTAGATTGCGTTTTAGAGGCTTTGGATAGAATGGATGAAGAACACCCAGGTCTTTTTCAAGCAGCCTCACAGAATGCTATGGAGGCCCTAATGGTCACAACTGTAGACAAATTAACCCAGGGGAGACAGACTTTTGATTGGACAGTATGCAGAAACCAACCTGCTGCAACGGCACTGAACACAACAATAACCTCTTTTAGGTTGAATGATTTAAATGGAGCCGACAAAGGTGGATTAATACCTTTTTGCCAGGATATCATTGATTCATTAGACCGACCTGAAATGACTTTCTTCTCAGTAAAGAATATAAAGAAAAAATTGCCTGCCAAAAACAGAAAGGGTTTCCTCATAAAGAGGATACCAATGAAGGTAAAAGACAAAATAACCAAAGTGGAATACATCAAAAGAGCATTATCATTAAACACAATGACAAAAGACGCTGAAAGAGGCAAACTGAAAAGAAGAGCGATTGCCACTGCTGGAATACAAATCAGAGGGTTTGTATTAGTAGTTGAAAACTTGGCTAAAAATATATGTGAAAATCTAGAACAAAGTGGTTTACCAGTAGGTGGAAACGAGAAGAAAGCCAAACTGTCAAACGCAGTGGCCAAAATGCTCAGTAACTGCCCACCAGGAGGGATTAGCATGACAGTAACAGGAGACAATACAAAATGGAATGAATGTTTAAACCCAAGAATCTTTTTGGCTATGACTGAAAGAATAACCAGAGACAGCCCAGTTTGGTTCAGGGATTTTTGTAGTATAGCACCGGTCCTGTTCTCCAATAAGATAGCAAGATTGGGGAAAGGGTTTATGATAACAAGCAAAACAAAAAGACTGAAGGCTCAAATACCTTGTCCTGATCTGTTTAGTATACCGTTAGAAAGATATAATGAAGAAACAAGGGCAAAATTGAAAAAGCTAAAACCATTCTTCAATGAAGAAGGAACTGCATCTTTGTCGCCTGGGATGATGATGGGAATGTTTAATATGCTATCTACCGTGTTGGGAGTAGCTGCACTAGGTATCAAGAACATTGGAAACAAAGAATACTTATGGGATGGACTGCAATCTTCTGATGATTTTGCTCTGTTTGTTAATGCAAAGGATGAAGAAACATGTATGGAAGGAATAAACGACTTTTACCGAACATGTAAATTATTGGGAGTAAACATGAGCAAAAAGAAAAGTTACTGTAATGAGACTGGAATGTTTGAATTTACAAGCATGTTCTACAGAGATGGATTTGTATCTAATTTTGCAATGGAACTCCCTTCGTTTGGGGTTGCTGGAGTAAATGAATCAGCAGATATGGCAATAGGAATGACAATAATAAAGAACAACATGATCAACAATGGAATGGGTCCGGCAACAGCACAAACAGCCATACAGTTATTCATAGCTGATTATAGATACACCTACAAATGCCACAGGGGAGATTCCAAAGTAGAAGGAAAGAGAATGAAAATCATAAAGGAGTTATGGGAAAACACTAAAGGAAGAGATGGTCTATTAGTAGCAGATGGTGGGCCCAACATTTACAATTTGAGAAACCTGCATATCCCAGAAATAGTATTAAAGTATAATCTAATGGACCCTGAATACAAAGGGCGGTTACTTCATCCTCAAAATCCCTTTGTGGGACATTTGTCTATTGAGGGCATCAAAGAGGCAGACATAACCCCAGCACATGGTCCAGTAAAGAAAATGGACTACGATGCGGTGTCTGGAACTCATAGTTGGAGAACCAAAAGAAACAGATCTATACTAAACACTGATCAGAGGAACATGATTCTTGAGGAACAATGCTACGCTAAATGTTGCAACCTATTTGAGGCCTGTTTTAACAGTGCATCATACAGGAAGCCAGTGGGTCAACATAGCATGCTTGAGGCTATGGCCCACAGATTAAGAATGGATGCACGATTAGATTATGAATCAGGGAGAATGTCAAAGGATGATTTTGAGAAAGCAATGGCTCACCTTGGTGAGATTGGGTACATATAAGCTTCGAAGATGTTTATGGGGTTATTGGTCATCATTGAATACATGCGATACACAAATGATTAAAATGAAAAAAGGCTCGTGTTTCTACT(PB2, B/Brisbane/60/08) SEQ ID NO: 13AGCAGAAGCGGAGCGTTTTCAAGATGACATTGGCCAAAATTGAATTGTTAAAACAACTGCTAAGGGACAATGAAGCCAAAACAGTTTTGAAGCAAACAACGGTAGACCAATATAACATAATAAGAAAATTCAATACATCAAGGATTGAAAAGAATCCTTCACTAAGGATGAAGTGGGCCATGTGTTCTAATTTTCCCTTGGCTCTAACCAAGGGCGATATGGCAAACAGAATCCCCTTGGAATACAAAGGGATACAACTTAAAACAAATGCTGAAGACATAGGAACCAAAGGCCAAATGTGCTCAATAGCAGCAGTTACTTGGTGGAATACATATGGACCAATAGGAGATACTGAAGGTTTCGAAAGGGTCTACGAAAGCTTTTTTCTCAGAAAAATGAGACTTGACAACGCCACTTGGGGCCGAATAACTTTTGGCCCAGTTGAAAGAGTGAGAAAAAGGGTACTGCTAAACCCTCTCACCAAGGAAATGCCTCCGGATGAGGCGAGCAATGTGATAATGGAAATATTGTTCCCTAAAGAAGCAGGAATACCAAGAGAATCCACTTGGATACATAGGGAACTGATAAAAGAAAAAAGAGAAAAATTGAAAGGAACAATGATAACTCCAATCGTACTGGCATACATGCTTGAAAGAGAACTGGTTGCTCGAAGAAGATTCTTGCCAGTGGCAGGAGCAACATCAGCTGAGTTCATAGAAATGCTACACTGCTTACAAGGTGAAAATTGGAGACAAATATATCACCCAGGAGGGAATAAATTAACTGAGTCCAGGTCTCAATCAATGATAGTAGCTTGTAGAAAAATAATCAGAAGATCAATAGTCGCTTCAAACCCACTGGAGCTAGCTGTAGAAATTGCAAACAAGACTGTGATAGATACTGAACCTTTAAAGTCATGTCTGGCAGCCATAGACGGAGGTGATGTAGCTTGTGACATAATAAGAGCTGCATTAGGACTAAAGATCAGACAAAGACAAAGATTTGGACGGCTTGAGCTAAAAAGAATATCAGGAAGAGGATTCAAAAATGATGAAGAAATATTAATAGGGAACGGAACAATACAGAAGATTGGAATATGGGACGGGGAAGAGGAGTTCCATGTAAGATGTGGTGAATGCAGGGGAATATTAAAAAAGAGTAAAATGAAACTGGAAAAACTACTGATAAATTCAGCCAAAAAGGAGGATATGAGAGATTTAATAATCTTATGCATGGTATTTTCTCAAGACACTAGGATGTTCCAAGGAGTGAGAGGAGAAATAAATTTTCTTAATCGAGCAGGCCAACTTTTATCTCCAATGTACCAACTCCAACGATATTTTTTGAATAGAAGCAACGACCTTTTTGATCAATGGGGGTATGAGGAATCACCCAAAGCAAGTGAACTACATGGGATAAATGAATCAATGAATGCATCTGACTATACATTGAAAGGGATTGTAGTGACAAGAAATGTAATTGACGACTTTAGCTCTATTGAAACAGAAAAAGTATCCATAACAAAAAATCTTAGTTTAATAAAAAGGACTGGGGAAGTCATAATGGGAGCTAATGACGTGAGTGAATTAGAATCACAAGCACAGCTGATGATAACATATGATACACCTAAAATGTGGGAAATGGGAACAACCAAAGAACTGGTGCAAAACACTTATCAATGGGTGCTAAAAAACTTGGTGACACTGAAGGCTCAGTTTCTTCTAGGAAAAGAGGACATGTTCCAATGGGATGCATTTGAAGCATTTGAGAGCATAATTCCTCAGAAGATGGCTGGTCAGTACAGTGGATTTGCAAGAGCAGTGCTCAAACAAATGAGAGACCAGGAGGTTATGAAAACTGACCAGTTCATAAAGTTGTTGCCTTTTTGTTTCTCACCACCAAAATTAAGGAGCAATGGGGAGCCTTATCAATTCTTAAAACTTGTGTTGAAAGGAGGAGGGGAAAATTTCATCGAAGTAAGGAAAGGGTCCCCTCTATTTTCCTATAATCCACAAACAGAAGTCCTAACTATATGCGGCAGAATGATGTCATTAAAAGGGAAAATTGAAGATGAAGAAAGGAATAGATCAATGGGTAATGCAGTATTAGCAGGCTTTCTCGTTAGTGGCAAGTATGACCCAGATCTTGGAGATTTCAAAACTATTGAAGAACTTGAAAAGCTGAAACCGGGGGAAAAGGCAAACATCTTACTTTATCAAGGAAAACCAGTTAAAGTAGTTAAAAGGAAAAGGTATAGTGCTTTGTCCAATGACATTTCACAAGGAATTAAGAGACAAAGAATGACAGTTGAGTCTATGGGGTGGGCCTTGAGCTAATATAAATTTATCCATTAATTCAATGAACGCAATTGAGTGAAAAATGCTCGTGTTTCTACT (NP, B/Brisbane/60/08) SEQ ID NO: 14AGCAGAAGCACAGCATTTTCTTGTGAACTTCAAGCACCAGTAAAAGAACTGAAAATCAAAATGTCCAACATGGATATTGACGGTATAAACACTGGGACAATTGACAAAACACCGGAAGAAATAACTTCTGGAACCAGTGGGACAACCAGACCAATCATTAGACCAGCAACCCTTGCCCCACCAAGCAACAAACGAACCCGTAACCCATCCCCGGAAAGAGCAACCACAAGCAGTGAAGATGATGTCGGAAGGAAAACCCAAAAGAAACAGACCCCGACAGAGATAAAGAAGAGCGTCTACAACATGGTGGTGAAACTGGGCGAATTCTATAACCAGATGATGGTCAAAGCTGGACTCAATGATGACATGGAGAGAAATCTAATCCAAAATGCGCATGCCGTGGAAAGAATTCTATTGGCTGCCACTGATGACAAGAAAACCGAGTTCCAGAAGAAAAAGAATGCCAGAGATGTCAAAGAAGGGAAAGAAGAAATAGATCACAACAAAACAGGAGGCACCTTTTACAAGATGGTAAGAGATGATAAAACCATCTACTTCAGCCCTATAAGAATTACCTTTTTAAAAGAAGAGGTGAAAACAATGTACAAAACCACCATGGGGAGTGATGGCTTCAGTGGACTAAATCACATAATGATTGGGCATTCACAGATGAATGATGTCTGTTTCCAAAGATCAAAGGCACTAAAAAGAGTTGGACTTGATCCTTCATTAATCAGTACCTTTGCGGGAAGCACAGTCCCCAGAAGATCAGGTGCGACTGGTGTTGCAATCAAAGGAGGTGGAACCTTAGTGGCTGAAGCCATTCGATTTATAGGAAGAGCAATGGCAGACAGAGGGCTATTGAGAGACATCAAAGCCAAGACTGCCTATGAAAAGATTCTTCTGAATCTAAAAAACAAATGCTCTGCGCCCCAACAAAAGGCTCTAGTTGATCAAGTGATCGGAAGCAGAAATCCGGGGATTGCAGACATTGAAGATCTAACCCTGCTTGCTCGTAGTATGGTCGTTGTTAGGCCCTCTGTGGCAAGCAAAGTGGTGCTTCCCATAAGCATTTACGCCAAAATACCTCAACTAGGGTTCAATGTTGAAGAGTACTCTATGGTTGGGTACGAAGCCATGGCTCTTTACAATATGGCAACACCTGTGTCCATATTAAGAATGGGAGATGATGCAAAAGATAAATCGCAATTATTCTTCATGTCTTGCTTCGGAGCTGCCTATGAAGACCTGAGAGTTTTGTCTGCATTAACAGGCACAGAATTCAAGCCTAGATCAGCATTAAAATGCAAGGGTTTCCATGTTCCAGCAAAGGAACAGGTAGAAGGAATGGGAGCAGCTCTGATGTCCATCAAGCTCCAGTTTTGGGCTCCGATGACCAGATCTGGGGGGAACGAAGTAGGTGGAGACGGAGGGTCTGGCCAAATAAGCTGCAGCCCAGTGTTTGCAGTGGAAAGACCTATTGCTCTAAGCAAGCAAGCTGTAAGAAGAATGCTGTCAATGAATATTGAGGGACGTGATGCAGATGTCAAAGGAAATCTACTCAAGATGATGAATGACTCAATGGCTAAGAAAACCAGTGGAAATGCTTTCATTGGGAAGAAAATGTTTCAAATATCAGACAAAAACAAAACCAATCCCATTGAAATTCCAATTAAGCAGACCATCCCCAATTTCTTCTTTGGGAGGGACACAGCAGAGGATTATGATGACCTCGATTATTAAGGCAACAAAATAGACACTATGACTGTGATTGTTTCAATACGTTTGGAATGTGGGTGTTTATTCTTATTAAAATAAATATAAAAAATGCTGTTGTTTCTACT(M, B/Brisbane/60/08) SEQ ID NO: 15AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCATTGACAGAAGATGGAGAAGGCAAAGCAGAACTAGCAGAAAAATTACACTGTTGGTTTGGTGGGAAAGAATTTGACCTAGACTCTGCCTTGGAATGGATAAAAAACAAAAGATGCTTAACTGATATACAAAAAGCACTAATTGGTGCCTCTATATGCTTTTTAAAACCCAAAGACCAGGAAAGAAAAAGAAGATTCATCACAGAGCCCTTATCAGGAATGGGAACAACAGCAACAAAAAAGAAAGGCCTGATTCTGGCTGAGAGAAAAATGAGAAGATGTGTGAGCTTTCATGAAGCATTTGAAATAGCAGAAGGCCATGAAAGCTCAGCGCTACTATACTGTCTCATGGTCATGTACCTGAATCCTGGAAATTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTATGCGAGAAACAAGCATCACATTCACACAGGGCTCATAGCAGAGCAGCGAGATCTTCAGTGCCTGGAGTGAGACGAGAAATGCAGATGGTCTCAGCTATGAACACAGCAAAAACAATGAATGGAATGGGAAAAGGAGAAGACGTCCAAAAGCTGGCAGAAGAGTTGCAAAGCAACATTGGAGTGCTGAGATCTCTTGGGGCAAGCCAAAAGAATGGGGAAGGGATTGCAAAGGATGTAATGGAAGTGCTAAAGCAGAGCTCCATGGGAAATTCAGCTCTTGTGAAGAAATATCTATAATGCTCGAACCATTTCAGATTCTTACAATTTGTTCTTTTATCTTATCAGCTCTCCATTTCATGGCTTGGACAATAGGGCATTTGAATCAAATAAAAAGAGGAATAAACATGAAAATACGAATAAAAGGTCCAAACAAAGAGACAATAAACAGAGAGGTATCAATTTTGAGACACAGTTACCAAAAAGAAATCCAGGCCAAAGAAACAATGAAGGAAGTACTCTCTGACAACATGGAGGTATTGAATGACCACATAATAATTGAGGGGCTTTCTGCCGAAGAGATAATAAAAATGGGTGAAACAGTTTTGGAGATAGAAGAATTGCATTAAATTCAATTTTACTGTATTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGCAAATAAACTGGAAAAAGTGCGTTGTTTCTACT (NS, B/Brisbane/60/08) SEQ ID NO: 16AGCAGAAGCAGAGGATTTGTTTAGTCACTGGCAAACAGGGAAAAATGGCGAACAACAACATGACCACAACACAAATTGAGGTGGGTCCGGGAGCAACCAATGCCACCATAAACTTTGAAGCAGGAATTCTAGAGTGCTATGAAAGGCTTTCATGGCAAAGAGCCCTTGACTACCCTGGTCAAGACCGCCTAAACAGACTAAAGAGAAAATTAGAGTCAAGAATAAAGACTCACAACAAAAGTGAGCCTGAAAGTAAAAGGATGTCCCTTGAAGAGAGAAAAGCAATTGGAGTAAAAATGATGAAAGTACTCCTATTTATGAATCCGTCTGCTGGAATTGAAGGGTTTGAGCCATACTGTATGAAAAGTTCCTCAAATAGCAACTGTACGAAATACAATTGGACTGATTACCCTTCAACACCAGAGAGGTGCCTTGATGACATAGAGGAAGAACCAGAGGATGTTGATGGCCCAACTGAAATAGTATTAAGGGACATGAACAACAAAGATGCAAGGCAAAAGATAAAGGAGGAAGTAAACACTCAGAAAGAAGGGAAGTTCCGTTTGACAATAAAAAGGGATATGCGTAATGTATTGTCCTTGAGAGTGTTGGTAAACGGAACATTCCTCAAACACCCCAATGGACACAAGTCCTTATCAACTCTGCATAGATTGAATGCATATGACCAGAGTGGAAGGCTTGTTGCTAAACTTGTTGCCACTGATGATCTTACAGTGGAGGATGAAGAAGATGGCCATCGGATCCTCAACTCACTCTTCGAGCGTCTTAATGAAGGACATTCAAAGCCAATTCGAGCAGCTGAAACTGCGGTGGGAGTCTTATCCCAATTTGGTCAAGAGCACCGATTATCACCAGAAGAGGGAGACAATTAGACTGGTCACGGAAGAACTTTATCTTTTAAGTAAAAGAATTGATGATAACATACTATTCCACAAAACAGTAATAGCTAACAGCTCCATAATAGCTGACATGGTTGTATCATTATCATTATTAGAAACATTGTATGAAATGAAGGATGTGGTTGAAGTGTACAGCAGGCAGTGCTTGTGAATTTAAAATAAAAATCCTCTTGTTACTACT (HA, B/Brisbane/60/08) SEQ ID NO: 17AGCAGAAGCAGAGCATTTTCTAATATCCACAAAATGAAGGCAATAATTGTACTACTCATGGTAGTAACATCCAATGCAGATCGAATCTGCACTGGGATAACATCGTCAAACTCACCACATGTCGTCAAAACTGCTACTCAAGGGGAGGTCAATGTGACTGGTGTAATACCACTGACAACAACACCCACCAAATCTCATTTTGCAAATCTCAAAGGAACAGAAACCAGGGGGAAACTATGCCCAAAATGCCTCAACTGCACAGATCTGGACGTAGCCTTGGGCAGACCAAAATGCACGGGGAAAATACCCTCGGCAAGAGTTTCAATACTCCATGAAGTCAGACCTGTTACATCTGGGTGCTTTCCTATAATGCACGACAGAACAAAAATTAGACAGCTGCCTAACCTTCTCCGAGGATACGAACATATCAGGTTATCAACCCATAACGTTATCAATGCAGAAAATGCACCAGGAGGACCCTACAAAATTGGAACCTCAGGGTCTTGCCCTAACATTACCAATGGAAACGGATTTTTCGCAACAATGGCTTGGGCCGTCCCAAAAAACGACAAAAACAAAACAGCAACAAATCCATTAACAATAGAAGTACCATACATTTGTACAGAAGGAGAAGACCAAATTACCGTTTGGGGGTTCCACTCTGACAACGAGGCCCAAATGGCAAAGCTCTATGGGGACTCAAAGCCCCAGAAGTTCACCTCATCTGCCAACGGAGTGACCACACATTACGTTTCACAGATTGGTGGCTTCCCAAATCAAACAGAAGACGGAGGACTACCACAAAGTGGTAGAATTGTTGTTGATTACATGGTGCAAAAATCTGGGAAAACAGGAACAATTACCTATCAAAGGGGTATTTTATTGCCTCAAAAGGTGTGGTGCGCAAGTGGCAGGAGCAAGGTAATAAAAGGATCCTTGCCTTTAATTGGAGAAGCAGATTGCCTCCACGAAAAATACGGTGGATTAAACAAAAGCAAGCCTTACTACACAGGGGAACATGCAAAGGCCATAGGAAATTGCCCAATATGGGTGAAAACACCCTTGAAGCTGGCCAATGGAACCAAATATAGACCTCCTGCAAAACTATTAAAGGAAAGGGGTTTCTTCGGAGCTATTGCTGGTTTCTTAGAAGGAGGATGGGAAGGAATGATTGCAGGTTGGCACGGATACACATCCCATGGGGCACATGGAGTAGCGGTGGCAGCAGACCTTAAGAGCACTCAAGAGGCCATAAACAAGATAACAAAAAATCTCAACTCTTTGAGTGAGCTGGAAGTAAAGAATCTTCAAAGACTAAGCGGTGCCATGGATGAACTCCACAACGAAATACTAGAACTAGATGAGAAAGTGGATGATCTCAGAGCTGATACAATAAGCTCACAAATAGAACTCGCAGTCCTGCTTTCCAATGAAGGAATAATAAACAGTGAAGATGAACATCTCTTGGCGCTTGAAAGAAAGCTGAAGAAAATGCTGGGCCCCTCTGCTGTAGAGATAGGGAATGGATGCTTTGAAACCAAACACAAGTGCAACCAGACCTGTCTCGACAGAATAGCTGCTGGTACCTTTGATGCAGGAGAATTTTCTCTCCCCACCTTTGATTCACTGAATATTACTGCTGCATCTTTAAATGACGATGGATTGGATAATCATACTATACTGCTTTACTACTCAACTGCTGCCTCCAGTTTGGCTGTAACACTGATGATAGCTATCTTTGTTGTTTATATGGTCTCCAGAGACAATGTTTCTTGCTCCATCTGTCTATAAGGGAAGTTAAGCCCTGTATTTTCCTTTATTGTAGTGCTTGTTTACTTGTTGTCATTACAAAGAAACGTTATTGAAAAATGCTCTTGTTACTACT (NA, B/Brisbane/60/08)SEQ ID NO: 18AGCAGAAGCAGAGCATCTTCTCAAAACTGAAGCAAATAGGCCAAAAATGAACAATGCTACCTTCAACTATACAAACGTTAACCCTATTTCTCACATCAGGGGGAGTATTATTATCACTATATGTGTCAGCTTCATTATCATACTTACTATATTCGGATATATTGCTAAAATTCTCACCAACAGAAATAACTGCACCAACAATGCCATTGGATTGTGCAAACGCATCAAATGTTCAGGCTGTGAACCGTTCTGCAACAAAAGGGGTGACACTTCTTCTCCCAGAACCGGAGTGGACATACCCGCGTTTATCTTGCCCGGGCTCAACCTTTCAGAAAGCACTCCTAATTAGCCCTCATAGATTCGGAGAAACCAAAGGAAACTCAGCTCCCTTGATAATAAGGGAACCTTTTATTGCTTGTGGACCAAATGAATGCAAACACTTTGCTCTAACCCATTATGCAGCCCAACCAGGGGGATACTACAATGGAACAAGAGGAGACAGAAACAAGCTGAGGCATCTAATTTCAGTCAAATTGGGCAAAATCCCAACAGTAGAAAACTCCATTTTCCACATGGCAGCATGGAGCGGGTCCGCGTGCCATGATGGTAAGGAATGGACATATATCGGAGTTGATGGCCCTGACAATAATGCATTGCTCAAAGTAAAATATGGAGAAGCATATACTGACACATACCATTCCTATGCAAACAAAATCCTAAGAACACAAGAAAGTGCCTGCAATTGCATCGGGGGAAATTGTTATCTTATGATAACTGATGGCTCAGCTTCAGGTGTTAGTGAATGCAGATTTCTTAAGATTCGAGAGGGCCGAATAATAAAAGAAATATTTCCAACAGGAAGAGTAAAACACACTGAGGAATGCACATGCGGATTTGCCAGCAATAAAACCATAGAATGTGCCTGTAGAGATAACAGTTACACAGCAAAAAGACCTTTTGTCAAATTAAACGTGGAGACTGATACAGCAGAAATAAGATTGATGTGCACAGATACTTATTTGGACACCCCCAGACCAAACGATGGAAGCATAACAGGCCCTTGTGAATCTAATGGGGACAAAGGGAGTGGAGGCATCAAGGGAGGATTTGTTCATCAAAGAATGGAATCCAAGATTGGAAGGTGGTACTCTCGAACGATGTCTAAAACTGAAAGGATGGGGATGGGACTGTATGTCAAGTATGATGGAGACCCATGGGCTGACAGTGATGCCCTAGCTTTTAGTGGAGTAATGGTTTCAATGAAAGAACCTGGTTGGTACTCCTTTGGCTTCGAAATAAAAGATAAGAAATGCGATGTCCCCTGTATTGGGATAGAGATGGTACATGATGGTGGAAAAGAGACTTGGCACTCAGCAGCAACAGCCATTTACTGTTTAATGGGCTCAGGACAGCTGCTGTGGGACACTGTCACAGGTGTTGACATGGCTCTGTAATGGAGGAATGGTTGAGTCTGTTCTAAACCCTTTGTTCCTGTTTTGTTTGAACAATTGTCCTTACTAAACTTAATTGTTTCTGAAAAATGCTCTTGTTACTACT (NP, B/Lee/40) SEQ ID NO: 19MSNMDIDSINTGTIDKKPEELTPGTSGATRPIIKPATLAPPSNKRTRNPSPERTTTSSETDIGRKIQKKQTPTEIKKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAQAVERILLAATDDKKTEYQKKRNARDVKEGKEEIDHNKTGGTFYKMVRDDKTIYFSPIKITFLKEEVKTMYKTTMGSDGFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTLPRRSGTTGVAIKGGGTLVAEAIRFIGRAMADRGLLRDIKAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRNPGIADIEDLTLLARSMIVVRPSVASKVVLPISIYAKIPQLGFNIEEYSMVGYEAMALYNMATPVSILRMGDDAKDKSQLFFMSCFGAAYEDLRVLSALTGTEFKPRSALKCKGFHVPAKEQVEGMGAALMSIKLQFWAPMTRSGGNEVSGEGGSGQISCSPVFAVERPIALSKQAVRRMLSMNVEGRDADVKGNLLKMMNDSMAKKTSGNAFIGKKMFQISDKNKVNPIEIPIKQTIPSFFFGRDTAEDYDDLDY (PA, B/Panama/45/90) SEQ ID NO: 20MDTFITRNFQTTIIQKAKNTMAEFSEDPELQPAMLFNICVHLEVCYVISDMNFLDEEGKSYTALEGQGKEQNLRPQYEVIEGMPRTIAWMVQRSLAQEHGIETPKYLADLFDYKTKRFIEVGITKGLADDYFWKKKEKLGNSMELMIFSYNQDYSLSNESSLDEEGKGRVLSRLTELQAELSLKNLWQVLIGEEDVEKGIDFKLGQTISRLRDISVPAGFSNFEGMRSYIDNIDPKGAIERNLARMSPLVSATPKKLKWEDLRPIGPHIYNHELPEVPYNAFLLMSDELGLANMTEGKSKKPKTLAKECLEKYSTLRDQTDPILIMKSEKANENFLWKLWRDCVNTISNEEMSNELQKTNYAKWATGDGLTYQKIMKEVAIDDETMCQEEPKIPNKCRVAAWVQTEMNLLSTLTSKRALDLPEIGPDVAPVEHVGSERRKYFVNEINCCKASTVMMKYVLFHTSLLNESNASMGKYKVIPITNRVVNEKGESFDMLYGLAVKGQSHLRGDTDVVTVVTFEFSGTDPRVDSGKWPKYTVFRIGSLFVSGREKSVYLYCRVNGTNKIQMKWGMEARRCLLQSMQQMEAIVEQESSIQGYDMTKACFKGDRVNSPKTFSIGTQEGKLVKGSFGKALRVIFTKCLMHYVFGNAQLEGFSAESRRLLLLIQALKDRKGPWVFDLEGMYSGIEECISNNPWVIQSAYWFNEWLGFEKEGSKVLESVDEIMNE (PB1, B/Panama/45/90) SEQ ID NO: 21MNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTGTGHTIDTVIRTHEYSNKGKQYVSDITGCTMVDPTNGPLPEDNEPSAYAQLDCVLEALDRMDEEHPGLFQAASQNAMEALMVTTVDKLTQGRQTFDWTVCRNQPAATALNTTITSFRLNDLNGADKGGLVPFCQDIIDSLDKPEMTFFSVKNIKKKLPAKNRKGFLIKRIPMKVKDRITRVEYIKRALSLNTMTKDAERGKLKRRAIATAGIQIRGFVLVVENLAKNICENLEQSGLPVGGNEKKAKLSNAVAKMLSNCPPGGISMTVTGDNTKWNECLNPRIFLAMTERITRDSPIWFRDFCSIAPVLFSNKIARLGKGFMITSKTKRLKAQIPCPDLFSIPLERYNEETRAKLKKLKPFFNEEGTASLSPGMMMGMFNMLSTVLGVAALGIKNIGNKEYLWDGLQSSDDFALFVNAKDEETCMEGINDFYRTCKLLGINMSKKKSYCNETGMFEFTSMFYRDGFVSNFAMEIPSFGVAGVNESADMAIGMTIIKNNMINNGMGPATAQTAIQLFIADYRYTYKCHRGDSKVEGKRMKIIKELWENTKGRDGLLVADGGPNIYNLRNLHIPEIVLKYNLMDPEYKGRLLHPQNPFVGHLSIEGIKEADITPAHGPVKKMDYDAVSGTHSWRTKRNRSILNTDQRNMILEEQCYAKCCNLFEACFNSASYRKPVGQHSMLEAMAHRLRVDARLDYESGRMSKDDFEKAMAHLGEIGYI(PB2, B/Panama/45/90) SEQ ID NO: 22MTLAKIELLKQLLRDNEAKTVLKQTTVDQYNIIRKFNTSRIEKNPSLRMKWAMCSNFPLALTKGDMANRIPLEYKGIQLKTNAEDIGTKGQMCSIAAVTWWNTYGPIGDTEGFEKVYESFFLRKMRLDNATWGRITFGPVERVRKRVLLNPLTKEMPPDEASNVIMEILFPKEAGIPRESTWIHRELIKEKREKLKGTMITPIVLAYMLERELVARRRFLPVAGATSAEFIEMLHCLQGENWRQIYHPGGNKLTESRSQSMIVACRKIIRRSIVASNPLELAVEIANKTVIDTEPLKSCLTAIDGGDVACDIIRAALGLKIRQRQRFGRLELKRISGRGFKNDEEILIGNGTIQKIGIWDGEEEFHVRCGECRGILKKSKMRMEKLLINSAKKEDMKDLIILCMVFSQDTRMFQGVRGEINFLNRAGQLLSPMYQLQRYFLNRSNDLFDQWGYEESPKASELHGINELMNASDYTLKGVVVTKNVIDDFSSTETEKVSITKNLSLIKRTGEVIMGANDVSELESQAQLMITYDTPKMWEMGTTKELVQNTYQWVLKNLVTLKAQFLLGKEDMFQWDAFEAFESIIPQKMAGQYSGFARAVLKQMRDQEVMKTDQFIKLLPFCFSPPKLRRNGEPYQFLRLVLKGGGENFIEVRKGSPLFSYNPQTEVLTICGRMMSLKGKIEDEERNRSMGNAVLAGFLVSGKYDPDLGDFKTIEELEKLKPGEKANILLYQGKPVKVVKRKRYSALSNDISQGIKRQRMTVESMGWALS(NP, B/Panama/45/90) SEQ ID NO: 23MSNMDIDGINTGTIDKTPEEITSGTSGTTRPIIRPATLAPPSNKRTRNPSPERATTSSEADVGRKTQKKQTPTEIKKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAHAVERILLAATDDKKTEFQRKKNARDVKEGKEEIDHNKTGGTFYKMVRDDKTIYFSPIRITFLKEEVKTMYKTTMGSDGFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTLPRRSGATGVAIKGGGTLVAEAIRFIGRAMADRGLLRDIKAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRNPGIADIEDLTLLARSMVVVRPSVASKVVLPISIYAKIPQLGFNVEEYSMVGYEAMALYNMATPVSILRMGDDAKDKSQLFFMSCFGAAYEDLRVLSALTGIEFKPRSALKCKGFHVPAKEQVEGMGAALMSIKLQFWAPMTRSGGNEVGGDGGSGQISCSPVFAVERPIALSKQAVRRMLSMNIEGRDADVKGNLLKMMNDSMAKKTNGNAFIGKKMFQISDKNKTNPVEIPIKQTIPNEFFGRDTAEDYDDLDY (M₁, B/Panama/45/90) SEQ ID NO: 24MSLFGDTIAYLLSLTEDGEGKAELAEKLHCWFGGKEFDLDSALEWIKNKRCLTDIQKALIGASICFLKPKDQERKRRFITEPLSGMGTTATKKKGLILAERKMRRCVSFHEAFEIAEGHESSALLYCLMVMYLNPGNYSMQVKLGTLCALCEKQASHSHRAHSRAARSSVPGVRREMQMVSAMNTAKTMNGMGKGEDVQKLAEELQSNIGVLRSLGASQKNGEGIAKDVMEVLKQSSMGNSALVKKYL (M₂, B/Panama/45/90) SEQ ID NO: 25MLEPFQILSICSFILSALHFMAWTIGHLNQIKRGVNMKIRIKNPNKETINREVSILRHSYQKEIQAKETMKEVLSDNMEVLSDHIVIEGLSAEEIIKMGETVLEVEELH (NS₁, B/Panama/45/90) SEQ ID NO: 26MADNMTTTQIEVGPGATNATINFEAGILECYERLSWQRALDYPGQDRLNKLKRKLESRIKTHNKSEPESKRMSLEERKAIGVKMMKVLLFMNPSAGVEGFEPYCMKNPSNSNCPDCNWADYPPTPGKYLDGIEEEPENVGDSTEIVLRDMNNKDARQKIKEEVNTQKEGKFRLTIKRDIRNVLSLRVLVNGTFIKHPNGYKSLSTLHRLNAYDQSGRLVAKLVATDDLTVEDEEDGHRILNSLFERLNEGHSKPIRAAETAVGVLSQFGQEHRLSPEERDN (NS₂, B/Panama/45/90)SEQ ID NO: 27MADNMTTTQIEWRMKKMAIGSSTHSSSVLMKDIQSQFEQLKLRWESYPNLVKSTDYHQKRETIRLVTEELYLLSKRIDDNILFHKTVIANSSIIADMIVSLSLLETLYEMKDVVEVYSRQCL (HA, B/Panama/45/90)SEQ ID NO: 28MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPNCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVINAERAPGGPYRLGTSGSCPNVTSRDGFFATMAWAVPRDNKTATNPLTVEVPYICTKGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL (NA, B/Panama/45/90)SEQ ID NO: 29MLPSTIQTLTLFLTSGGVLLSLYVSASLSYLLYSDILLKFSPTEITAPTMPLDCANASNVQAVNRSATKEMTLLLPEPEWTYPRLSCPGSTFQKALLISPHRFGETRGNSAPLTIREPFIACGPKECKHFALTHYAAQPGGYYNGTREDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGREWTYIGVDGPDSNALIKIKYGEAYTDTYHSYANNILRTQESACNCIGGDCYLMITDGSASGISKCRFLKIREGRIIKEIFPTGRVEHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTETYLDTPRPDDGSITGPCESNGDKGRGGIKGGFVHQRMASKIGRWYSRTMSKTERMGMELYVKYDGDPWTDSEALAPSGVMVSMEEPGWYSFGFEIKDKKCDVPCIGIEMVHDGGKKTWHSAATAIYCLMGSGQLLWDTVTGVDMAL (PA, B/Panama/45/90) SEQ ID NO: 30AGCAGAAGCGGTGCGTTTGATTTGCCATAATGGATACTTTTATTACAAGAAACTTCCAGACTACAATAATACAAAAGGCCAAAAACACAATGGCAGAATTTAGTGAAGATCCTGAATTACAACCAGCAATGCTATTCAACATCTGCGTCCATCTAGAGGTTTGCTATGTAATAAGTGACATGAATTTTCTTGACGAAGAAGGAAAATCATATACAGCATTAGAAGGACAAGGAAAAGAACAAAACTTGAGACCACAATATGAAGTAATTGAGGGAATGCCAAGAACCATAGCATGGATGGTCCAAAGATCCTTAGCTCAAGAGCATGGAATAGAGACTCCAAAGTATCTGGCTGATTTGTTTGATTATAAAACCAAGAGATTTATAGAAGTTGGAATAACAAAAGGATTGGCTGATGATTACTTTTGGAAAAAGAAAGAAAAGCTGGGAAATAGCATGGAACTGATGATATTCAGCTACAATCAAGACTATTCGTTAAGTAATGAATCCTCATTGGATGAGGAAGGGAAAGGGAGAGTGCTAAGCAGACTCACAGAACTTCAGGCTGAATTAAGTCTGAAAAACCTATGGCAAGTTCTCATAGGAGAAGAAGATGTTGAAAAGGGAATTGACTTTAAACTTGGACAAACAATATCTAGACTAAGGGATATATCTGTTCCAGCTGGTTTCTCCAATTTTGAAGGAATGAGGAGCTACATAGACAATATAGATCCTAAAGGAGCAATAGAAAGAAATCTAGCAAGGATGTCTCCCTTAGTATCAGCCACACCTAAAAAGTTGAAATGGGAGGACCTAAGACCAATAGGGCCTCACATTTACAACCATGAGTTACCAGAAGTTCCATATAATGCCTTTCTTCTAATGTCTGATGAATTGGGGCTGGCCAATATGACTGAGGGAAAGTCCAAAAAACCGAAGACATTAGCCAAAGAATGTCTAGAAAAGTACTCAACACTACGGGATCAAACTGACCCAATATTAATAATGAAAAGCGAAAAAGCTAACGAAAATTTCCTATGGAAGCTGTGGAGGGACTGTGTAAATACAATAAGTAATGAGGAAATGAGTAACGAGTTACAGAAAACCAATTATGCCAAGTGGGCCACAGGAGATGGATTAACATACCAGAAAATAATGAAAGAAGTAGCAATAGATGACGAAACAATGTGCCAAGAAGAGCCTAAAATCCCTAACAAATGTAGAGTGGCTGCTTGGGTTCAAACAGAGATGAATTTATTGAGCACTCTGACAAGTAAAAGAGCTCTGGACCTACCAGAAATAGGGCCAGACGTAGCACCCGTGGAGCATGTAGGGAGTGAAAGAAGGAAATACTTTGTTAATGAAATCAACTGCTGTAAGGCCTCTACAGTTATGATGAAGTATGTGCTTTTTCACACTTCATTATTGAATGAAAGCAATGCCAGCATGGGAAAATATAAAGTAATACCAATAACCAATAGAGTAGTAAATGAAAAAGGAGAAAGTTTCGACATGCTTTATGGTCTGGCGGTTAAAGGACAATCTCATCTGAGGGGAGATACTGATGTTGTAACAGTTGTGACTTTCGAATTTAGTGGTACAGATCCCAGAGTGGACTCAGGAAAGTGGCCAAAATATACTGTGTTTAGGATTGGCTCCCTATTTGTGAGTGGGAGGGAAAAATCTGTGTACCTATATTGCCGAGTGAATGGCACAAATAAGATCCAAATGAAATGGGGAATGGAAGCTAGAAGATGTCTGCTTCAATCAATGCAACAAATGGAAGCAATTGTTGAACAAGAATCATCGATACAAGGATATGACATGACCAAAGCTTGTTTCAAGGGAGACAGAGTAAATAGCCCCAAAACTTTTAGTATTGGGACTCAAGAAGGAAAACTAGTAAAAGGATCCTTTGGGAAAGCACTAAGAGTAATATTTACCAAATGTTTGATGCACTATGTATTTGGAAATGCCCAATTGGAGGGGTTTAGTGCCGAGTCTAGGAGACTTCTACTGTTAATTCAAGCACTAAAGGACAGAAAGGGCCCTTGGGTGTTCGACTTAGAGGGAATGTATTCTGGAATAGAAGAATGTATTAGTAACAACCCTTGGGTAATACAGAGTGCATACTGGTTCAATGAATGGTTGGGCTTTGAAAAGGAGGGGAGTAAAGTATTAGAATCAGTAGATGAAATAATGAATGAATGAAAAAACATAGTACTCAATTTGGTACTATTTTGTTCATTATGTATCTAAACATCCAATAAAAAGAATCGAGAATCAAAAATGCACGTGTTTCTACT(PB1, B/Panama/45/90) SEQ ID NO: 31AGCAGAAGCGGAGCCTTTAAGATGAATATAAATCCTTATTTTCTCTTCATAGATGTACCCATACAGGCAGCAATTTCAACAACATTCCCATACACCGGTGTTCCCCCTTACTCCCATGGAACGGGAACAGGCCACACAATAGACACCGTGATCAGAACACATGAGTACTCGAACAAGGGAAAACAGTATGTTTCTGACATCACAGGATGTACAATGGTAGATCCAACAAATGGGCCATTACCCGAAGACAATGAGCCGAGTGCCTATGCACAATTAGATTGCGTTCTGGAGGCTTTGGATAGAATGGATGAAGAACATCCAGGTTTGTTTCAAGCAGCCTCACAGAATGCCATGGAGGCACTAATGGTCACAACTGTAGACAAATTAACCCAGGGGAGACAGACTTTTGATTGGACAGTATGCAGAAACCAGCCTGCTGCAACGGCACTAAACACAACAATAACCTCCTTTAGGTTGAATGATTTGAATGGAGCTGACAAGGGTGGATTGGTACCCTTTTGCCAAGATATCATTGATTCATTGGACAAACCTGAAATGACTTTCTTCTCAGTAAAGAATATAAAGAAAAAATTGCCTGCTAAAAACAGAAAGGGTTTCCTCATAAAGAGAATACCAATGAAAGTAAAAGACAGGATAACCAGAGTGGAATACATCAAAAGAGCATTATCATTAAACACAATGACAAAAGATGCTGAAAGGGGCAAACTAAAAAGAAGAGCGATTGCAACCGCTGGAATACAAATCAGAGGGTTTGTATTAGTAGTTGAAAACTTGGCTAAAAATATCTGTGAAAATCTAGAACAAAGTGGTTTGCCCGTAGGTGGAAATGAAAAGAAGGCCAAACTGTCAAATGCAGTGGCCAAAATGCTCAGTAACTGCCCACCAGGAGGGATCAGCATGACAGTAACAGGAGACAATACTAAATGGAATGAATGCTTAAATCCAAGAATCTTTTTGGCTATGACTGAAAGGATAACAAGAGACAGCCCAATTTGGTTCCGGGATTTTTGTAGTATAGCACCGGTCTTGTTCTCCAATAAAATAGCCAGATTGGGAAAAGGATTTATGATAACAAGCAAAACAAAAAGACTGAAGGCTCAAATACCTTGTCCAGATCTGTTTAGCATACCATTAGAAAGATATAATGAAGAAACAAGGGCAAAATTAAAAAAGCTGAAACCATTCTTCAATGAAGAAGGAACGGCATCTTTGTCGCCTGGGATGATGATGGGAATGTTTAATATGCTATCTACCGTGTTGGGAGTAGCCGCACTAGGTATCAAAAACATTGGAAACAAAGAATATTTATGGGATGGACTGCAATCTTCTGATGATTTTGCTCTGTTTGTTAATGCAAAAGATGAAGAGACATGTATGGAAGGAATAAACGACTTTTACCGAACATGTAAATTATTGGGAATAAACATGAGCAAAAAGAAAAGTTACTGTAATGAAACTGGAATGTTTGAATTTACAAGCATGTTCTATAGAGATGGATTTGTATCTAATTTTGCAATGGAAATTCCTTCATTTGGAGTTGCTGGAGTAAATGAATCAGCAGATATGGCAATAGGAATGACAATAATAAAGAACAATATGATCAACAATGGGATGGGTCCAGCAACAGCACAAACAGCCATACAATTATTCATAGCTGATTATAGGTACACCTACAAATGCCACAGGGGAGATTCCAAAGTGGAAGGAAAAAGAATGAAAATTATAAAGGAGCTATGGGAAAACACTAAAGGAAGAGATGGTCTGTTAGTGGCAGATGGTGGGCCCAACATTTACAATTTGAGAAACTTACATATCCCAGAAATAGTATTGAAGTACAACCTAATGGACCCTGAATACAAAGGGCGGTTACTTCATCCTCAAAATCCATTTGTAGGACATTTATCTATTGAGGGCATCAAAGAAGCAGATATAACCCCAGCACATGGTCCCGTAAAGAAAATGGATTATGATGCAGTATCTGGAACTCATAGTTGGAGAACCAAAAGGAACAGATCTATACTAAATACTGACCAGAGGAACATGATTCTTGAGGAACAATGCTACGCTAAGTGTTGCAACCTTTTTGAGGCCTGTTTTAATAGTGCATCATACAGGAAACCAGTAGGTCAGCACAGCATGCTTGAGGCTATGGCCCACAGATTAAGAGTGGATGCACGACTAGATTATGAATCAGGAAGAATGTCAAAGGATGATTTTGAGAAAGCAATGGCTCACCTTGGTGAGATTGGGTACATATAAGCTCCGAAGATGTCTATGGGGTTATTGGTCATCATTGAATACATGTGATAAACAAATGATTAAAATGAAAAAAGGCTCGTGTTTCTACT(PB2, B/Panama/45/90) SEQ ID NO: 32AGCAGAAGCGGAGCGTTTTCAAGATGACATTGGCTAAAATTGAATTGTTAAAACAACTGTTAAGGGACAATGAAGCCAAAACAGTATTGAAACAAACAACGGTAGACCAATATAACATAATAAGAAAATTCAATACATCAAGAATTGAAAAGAACCCTTCATTGAGGATGAAGTGGGCAATGTGTTCTAATTTTCCCTTGGCTCTGACCAAGGGTGATATGGCAAACAGAATCCCCTTGGAATACAAGGGAATACAACTTAAAACAAATGCTGAAGACATAGGAACTAAAGGCCAAATGTGCTCAATAGCAGCAGTTACCTGGTGGAATACATATGGACCAATAGGAGATACTGAAGGTTTCGAAAAGGTCTACGAAAGCTTTTTTCTCAGAAAGATGAGACTTGACAATGCCACTTGGGGCCGAATAACTTTTGGCCCAGTTGAAAGAGTAAGAAAAAGGGTACTGCTAAACCCTCTCACCAAGGAAATGCCTCCAGATGAAGCAAGTAATGTGATAATGGAAATATTGTTCCCTAAGGAAGCAGGAATACCAAGAGAATCTACTTGGATACATAGGGAACTGATAAAAGAAAAAAGAGAAAAATTGAAAGGAACAATGATAACTCCCATTGTACTGGCATACATGCTTGAGAGAGAATTGGTTGCCAGAAGAAGGTTCCTGCCGGTGGCAGGAGCAACATCAGCTGAGTTCATAGAAATGCTACACTGCTTACAAGGTGAAAATTGGAGACAAATATATCACCCAGGAGGAAATAAACTAACTGAATCTAGGTCTCAATCGATGATTGTAGCTTGTAGAAAGATAATCAGAAGATCAATAGTCGCATCAAACCCATTAGAGCTAGCTGTAGAAATTGCAAACAAGACTGTGATAGATACTGAACCTTTAAAATCATGTCTGACAGCCATAGACGGAGGTGATGTAGCCTGTGACATAATAAGAGCTGCATTAGGACTAAAGATCAGACAAAGACAAAGATTTGGACGACTTGAACTAAAGAGAATATCAGGAAGAGGATTCAAAAATGATGAAGAAATATTAATCGGGAACGGAACAATACAGAAGATTGGAATATGGGACGGAGAAGAGGAGTTCCATGTAAGATGTGGTGAATGCAGGGGAATATTAAAAAAGAGCAAAATGAGAATGGAAAAACTACTAATAAATTCAGCTAAAAAGGAAGACATGAAAGATTTAATAATCTTGTGCATGGTATTTTCTCAAGACACTAGGATGTTCCAAGGAGTGAGAGGAGAAATAAATTTTCTTAATAGAGCAGGCCAACTTTTATCTCCAATGTACCAACTCCAAAGATATTTTTTGAATAGAAGCAACGATCTCTTTGATCAATGGGGGTATGAGGAATCACCCAAAGCAAGTGAGCTACATGGAATAAATGAATTAATGAATGCATCTGACTACACTTTGAAAGGGGTTGTAGTAACAAAAAATGTAATTGATGATTTTAGTTCTACTGAAACAGAAAAAGTATCTATAACAAAAAATCTTAGTTTAATAAAAAGGACTGGGGAAGTCATAATGGGGGCTAATGACGTAAGTGAATTAGAATCACAAGCTCAGCTAATGATAACATATGATACACCTAAGATGTGGGAGATGGGAACAACCAAAGAACTGGTGCAAAACACCTACCAATGGGTGCTGAAAAATTTGGTAACACTGAAGGCTCAGTTTCTTCTAGGAAAAGAAGACATGTTCCAATGGGATGCATTTGAAGCATTTGAAAGCATAATCCCCCAGAAGATGGCTGGCCAGTACAGTGGATTTGCAAGAGCAGTGCTCAAACAAATGAGAGACCAAGAGGTTATGAAAACTGACCAGTTCATAAAGTTGTTGCCCTTTTGTTTCTCACCACCAAAATTAAGGAGAAATGGGGAGCCTTATCAGTTCTTGAGGCTTGTATTGAAGGGAGGAGGAGAAAATTTCATCGAAGTAAGGAAAGGGTCCCCTCTATTCTCTTACAATCCACAAACAGAAGTCCTAACTATATGCGGCAGAATGATGTCATTAAAAGGGAAAATTGAAGATGAAGAAAGGAATAGATCAATGGGGAATGCAGTATTAGCGGGCTTTCTCGTTAGTGGCAAGTATGACCCAGATCTTGGAGATTTCAAAACTATTGAAGAACTTGAAAAGCTGAAACCGGGGGAGAAAGCAAACATCTTACTTTATCAAGGAAAGCCCGTTAAAGTAGTTAAAAGGAAAAGATATAGTGCTTTATCCAATGACATTTCACAAGGAATTAAGAGACAAAGAATGACAGTTGAGTCCATGGGGTGGGCCTTGAGCTAATATAAATTTATCCATTAATTCAATAAACACAATTGAGTGAAAAATGCTCGTGTTTCTACT (NP, B/Panama/45/90) SEQ ID NO: 33AGCAGAAGCACAGCATTTTCTTATTAACTTCAAGTACCAACAAAAGAACTGAAAATCAAAATGTCCAACATGGATATTGACGGTATCAACACTGGGACAATTGACAAAACACCGGAAGAAATAACTTCTGGAACCAGTGGGACAACCAGACCAATCATCAGACCAGCAACCCTTGCCCCACCAAGCAACAAACGAACCCGGAACCCATCCCCGGAAAGAGCAACCACAAGCAGTGAAGCTGATGTCGGAAGGAAAACCCAAAAGAAACAGACCCCGACAGAGATAAAGAAGAGCGTCTACAATATGGTAGTGAAACTGGGTGAATTCTATAACCAGATGATGGTCAAAGCTGGACTCAACGATGACATGGAGAGAAACCTAATCCAAAATGCGCATGCTGTGGAAAGAATTCTATTGGCTGCCACTGATGACAAGAAAACTGAATTCCAGAGGAAAAAGAATGCCAGAGATGTCAAAGAAGGAAAAGAAGAAATAGACCACAACAAAACAGGAGGCACCTTTTACAAGATGGTAAGAGATGATAAAACCATCTACTTCAGCCCTATAAGAATTACCTTTTTAAAAGAAGAGGTGAAAACAATGTACAAAACCACCATGGGGAGTGATGGCTTCAGTGGACTAAATCACATAATGATTGGGCATTCACAGATGAATGATGTCTGTTTCCAAAGATCAAAGGCCCTAAAAAGAGTTGGACTTGACCCTTCATTAATCAGTACCTTTGCAGGAAGCACACTCCCCAGAAGATCAGGTGCAACTGGTGTTGCAATCAAAGGAGGTGGAACTTTAGTGGCTGAAGCCATTCGATTTATAGGAAGAGCAATGGCAGACAGAGGGCTATTGAGAGACATCAAAGCCAAGACTGCCTATGAAAAGATTCTTCTGAATCTAAAAAACAAATGCTCTGCGCCCCAACAAAAGGCTCTAGTTGATCAAGTGATCGGAAGTAGAAATCCAGGGATTGCAGACATTGAAGACCTAACCCTGCTTGCTCGTAGTATGGTCGTTGTTAGGCCCTCTGTGGCGAGCAAAGTAGTGCTTCCCATAAGCATTTATGCTAAAATACCTCAACTAGGGTTCAATGTTGAAGAATACTCTATGGTTGGGTATGAAGCCATGGCTCTCTACAATATGGCAACACCTGTTTCCATATTAAGAATGGGAGATGATGCAAAAGATAAATCGCAATTATTCTTCATGTCTTGCTTCGGAGCTGCCTATGAAGACCTGAGAGTTTTGTCTGCATTAACAGGCATAGAATTCAAGCCTAGATCAGCATTAAAATGCAAGGGTTTCCATGTTCCAGCAAAGGAACAGGTGGAAGGAATGGGGGCAGCTCTGATGTCCATCAAGCTCCAGTTTTGGGCTCCAATGACCAGATCTGGAGGGAACGAAGTAGGTGGAGACGGAGGGTCTGGCCAAATAAGTTGCAGCCCAGTGTTTGCAGTAGAAAGACCTATTGCTCTAAGCAAGCAAGCTGTAAGAAGAATGCTTTCAATGAATATTGAGGGACGTGATGCAGATGTCAAAGGAAATCTACTCAAGATGATGAATGACTCAATGGCTAAGAAAACCAATGGAAATGCTTTCATTGGGAAGAAAATGTTTCAAATATCAGACAAAAACAAAACCAATCCCGTTGAAATTCCAATTAAGCAGACCATCCCCAATTTCTTCTTTGGGAGGGACACAGCAGAGGATTATGATGACCTCGATTATTAAAGCAACAAAATAGACACTATGACTGTGATTGTTTCAATACGTTTGGAATGTGGGTGTTTACTCTTATTGAAATAAATATAAAAAATGCTGTTGTTTCTACT(M, B/Panama/45/90) SEQ ID NO: 34AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCATTGACAGAAGATGGAGAAGGCAAAGCAGAACTAGCAGAAAAATTACACTGTTGGTTCGGTGGGAAAGAATTTGACCTAGACTCTGCCTTGGAATGGATAAAAAACAAAAGATGCTTAACTGATATACAGAAAGCACTAATTGGTGCCTCTATCTGCTTTTTAAAACCAAAAGACCAAGAAAGAAAAAGAAGATTCATCACAGAGCCCCTATCAGGAATGGGAACAACAGCAACAAAAAAGAAGGGCCTGATTCTAGCTGAGAGAAAAATGAGAAGATGTGTGAGTTTTCATGAAGCATTTGAAATAGCAGAAGGCCATGAAAGCTCAGCGCTACTATATTGTCTCATGGTCATGTACCTGAACCCTGGAAATTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTGTGCGAGAAACAAGCATCACATTCACACAGGGCTCATAGCAGAGCAGCAAGATCTTCAGTGCCTGGAGTGAGGCGAGAAATGCAGATGGTCTCAGCTATGAACACAGCAAAAACAATGAATGGAATGGGAAAGGGAGAAGACGTCCAAAAACTGGCAGAAGAGCTGCAAAGCAACATTGGAGTATTGAGATCTCTTGGGGCAAGTCAAAAGAATGGGGAAGGAATTGCAAAGGATGTGATGGAAGTGCTAAAGCAGAGCTCTATGGGAAATTCAGCTCTTGTGAAGAAATACCTATAATGCTCGAACCATTTCAGATTCTTTCAATTTGTTCTTTCATCTTATCAGCTCTCCATTTCATGGCTTGGACAATAGGGCATTTGAATCAAATAAAAAGAGGAGTAAACATGAAAATACGAATAAAAAATCCAAATAAAGAGACAATAAACAGAGAGGTATCAATTTTGAGACACAGTTACCAAAAAGAAATCCAGGCCAAAGAAACAATGAAGGAAGTACTCTCTGACAACATGGAGGTATTGAGTGACCACATAGTAATTGAGGGGCTTTCTGCTGAAGAGATAATAAAAATGGGTGAAACAGTTTTGGAGGTAGAAGAATTGCATTAAATTCAATTTTTACTGTATTTCTTGCTATGCATTTAAGCAAATTGTAATCAATGTCAGCAAATAAACTGGAAAAAGTGCGTTGTTTCTACT (NS, B/Panama/45/90) SEQ ID NO: 35AGCAGAAGCAGAGGATTTGTTTAGTCACTGGCAAACGAAAAAATGGCGGACAACATGACCACAACACAAATTGAGGTGGGTCCGGGAGCAACCAATGCCACCATAAACTTTGAAGCAGGAATTTTGGAGTGCTATGAAAGGCTTTCATGGCAAAGAGCCCTTGACTACCCTGGTCAAGACCGCCTAAACAAACTAAAGAGAAAATTGGAATCAAGAATAAAGACTCACAACAAAAGTGAGCCAGAAAGTAAAAGGATGTCTCTTGAAGAGAGAAAAGCTATTGGGGTAAAAATGATGAAAGTGCTCCTATTTATGAACCCATCTGCTGGAGTTGAAGGGTTTGAGCCATATTGTATGAAAAATCCCTCCAATAGCAACTGTCCAGACTGCAATTGGGCTGATTACCCTCCAACACCAGGAAAGTACCTTGATGGCATAGAAGAAGAACCGGAGAATGTTGGTGACTCAACTGAAATAGTATTAAGGGACATGAACAACAAAGATGCAAGGCAAAAGATAAAAGAGGAAGTAAACACTCAGAAAGAAGGGAAATTCCGTTTGACAATAAAAAGGGATATACGTAATGTGTTGTCCTTGAGAGTGTTGGTAAACGGAACATTCATCAAGCACCCTAATGGATACAAGTCCTTATCAACTCTGCATAGATTGAATGCATATGACCAGAGTGGAAGACTTGTTGCTAAACTTGTTGCTACTGATGATCTTACAGTGGAGGATGAAGAAGATGGCCATCGGATCCTCAACTCACTCTTCGAGCGTCTTAATGAAGGACATTCAAAGCCAATTCGAGCAGCTGAAACTGCGGTGGGAGTCTTATCCCAATTTGGTCAAGAGCACCGATTATCACCAGAAGAGAGAGACAATTAGACTGGTTACGGAAGAACTTTATCTTTTAAGTAAAAGAATTGATGATAACATATTGTTCCACAAAACAGTAATAGCCAACAGCTCCATAATAGCTGACATGATTGTATCATTATCATTATTGGAAACATTGTATGAAATGAAGGATGTGGTTGAAGTGTACAGCAGGCAGTGCTTGTGAATTTAAAATAAAAATCCTCTTGTTACTACT (NA, B/Panama/45/90) SEQ ID NO: 36AGCAGAAGCAGAGCATCTTCTCAAAACTGAGGCAAATAGGCCAAAAATGAACAATGCTACCTTCAACTATACAAACGTTAACCCTATTTCTCACATCAGGGGGAGTGTTATTATCACTATATGTGTCAGCTTCACTATCATACTTACTGTATTCGGATATATTGCTAAAATTTTCACCAACAGAAATAACTGCACCAACAATGCCATTGGATTGTGCAAACGCATCAAATGTTCAGGCTGTGAACCGTTCTGCAACAAAAGAGATGACACTTCTTCTCCCAGAACCGGAGTGGACATACCCTCGTTTATCTTGCCCGGGCTCAACCTTTCAGAAAGCACTCCTAATTAGCCCTCATAGATTCGGAGAAACCAGAGGAAACTCAGCTCCCTTGACAATAAGGGAACCTTTTATTGCTTGTGGACCAAAGGAATGCAAACACTTTGCTCTAACCCATTATGCAGCTCAACCAGGGGGATACTACAATGGAACAAGAGAGGACAGAAACAAGCTGAGGCATCTGATTTCAGTCAAATTGGGCAAAATACCAACAGTAGAAAACTCCATTTTCCACATGGCAGCTTGGAGCGGGTCCGCATGCCATGATGGTAGAGAATGGACATATATCGGAGTTGATGGCCCTGACAGTAATGCATTGATCAAAATAAAATATGGAGAAGCATATACTGACACATACCATTCCTATGCAAACAACATCCTAAGAACACAAGAAAGTGCCTGCAATTGCATTGGGGGAGATTGTTATCTTATGATAACTGATGGCTCAGCTTCAGGAATTAGTAAATGCAGATTTCTTAAGATTCGAGAGGGTCGAATAATAAAAGAAATATTTCCAACAGGAAGAGTAGAACATACTGAAGAATGCACATGCGGATTTGCCAGCAACAAAACCATAGAATGTGCCTGTAGAGATAACAGTTACACAGCAAAAAGACCCTTTGTCAAATTAAATGTGGAGACTGATACAGCTGAAATAAGATTGATGTGCACAGAGACTTATTTGGACACCCCCAGACCAGATGATGGAAGCATAACAGGGCCTTGCGAATCTAATGGGGACAAAGGGCGTGGAGGCATCAAGGGAGGATTTGTTCATCAAAGAATGGCATCCAAGATTGGAAGATGGTACTCTCGAACGATGTCTAAAACTGAAAGAATGGGGATGGAACTGTATGTCAAGTATGATGGAGACCCATGGACTGACAGTGAAGCCCTTGCTCCTAGTGGAGTAATGGTTTCAATGGAAGAACCTGGTTGGTATTCTTTTGGCTTCGAAATAAAAGATAAGAAATGTGATGTCCCCTGTATTGGGATAGAGATGGTACACGATGGTGGAAAAAAGACTTGGCACTCAGCAGCAACAGCCATTTACTGTTTAATGGGCTCAGGACAATTGCTATGGGACACTGTCACAGGTGTTGATATGGCTCTGTAATGGAGGAATGGTTGAGTCTGTTCTAAACCCTTTGTTCCTATTTTGTTTGAATAATTGTCCTTACTGAACTTAATTGTTTCTGAAAAATGCTCTTGTTACTACT (HA, B/Panama/45/90) SEQ ID NO: 37AGCAGAAGCAGAGCATTTTCTAATATCCACAAAATGAAGGCAATAATTGTACTACTCATGGTAGTAACATCCAACGCAGATCGAATCTGCACTGGGATAACATCTTCAAACTCACCTCATGTGGTCAAAACAGCTACTCAAGGGGAAGTCAATGTGACTGGTGTGATACCACTGACAACAACACCAACAAAATCTCATTTTGCAAATCTAAAAGGAACAAAGACCAGAGGGAAACTATGCCCAAACTGTCTCAACTGCACAGATCTGGATGTGGCCTTGGGCAGACCAATGTGTGTGGGGACCACACCTTCGGCAAAAGCTTCAATACTCCACGAAGTCAGACCTGTTACATCCGGGTGCTTTCCTATAATGCACGACAGAACAAAAATCAGACAGCTACCCAATCTTCTCAGAGGATATGAAAATATCAGATTATCAACCCAAAACGTTATCAACGCAGAAAGAGCACCAGGAGGACCCTACAGACTTGGAACCTCAGGATCTTGCCCTAACGTTACCAGTAGAGACGGATTCTTCGCAACAATGGCTTGGGCTGTCCCAAGGGACAACAAAACAGCAACGAATCCACTAACAGTAGAAGTACCATACATTTGTACAAAAGGAGAAGACCAAATTACTGTTTGGGGGTTCCATTCTGATGACAAAACCCAAATGAAAAACCTCTATGGAGACTCAAATCCTCAAAAGTTCACCTCATCTGCCAATGGAGTAACCACACATTATGTTTCTCAGATTGGTGGCTTCCCAAATCAAACAGAAGACGGAGGGCTACCACAAAGCGGCAGAATTGTTGTTGATTACATGGTGCAAAAACCTGGGAAAACAGGAACAATTGTCTATCAAAGAGGTGTTTTGTTGCCTCAAAAGGTGTGGTGCGCAAGTGGCAGGAGCAAGGTAATAAAAGGGTCCTTGCCTTTAATTGGTGAAGCAGATTGCCTTCACGAAAAATACGGTGGATTAAACAAAAGCAAGCCTTACTACACAGGAGAACATGCAAAAGCCATAGGAAATTGCCCAATATGGGTGAAAACACCTTTGAAGCTTGCCAATGGAACCAAATATAGACCTCCTGCAAAACTATTAAAGGAAAGGGGTTTCTTCGGAGCTATTGCTGGTTTCTTAGAAGGAGGATGGGAAGGAATGATTGCAGGTTGGCACGGATACACATCTCATGGAGCACATGGAGTGGCAGTGGCAGCAGACCTTAAGAGTACGCAAGAAGCCATAAACAAGATAACAAAAAATCTCAATTCTTTGAGTGAGCTAGAAGTAAAGAATCTTCAAAGACTAAGTGGTGCCATGGATGAACTCCACAACGAAATACTCGAGCTGGATGAGAAAGTGGATGATCTCAGAGCTGACACAATAAGCTCGCAAATAGAGCTTGCAGTCTTGCTTTCCAACGAAGGAATAATAAACAGTGAAGATGAGCATCTATTGGCACTTGAGAGAAAACTAAAGAAAATGCTGGGTCCCTCTGCTGTAGACATAGGGAATGGATGCTTCGAAACCAAACACAAGTGCAACCAGACCTGCTTAGACAGAATAGCTGCTGGCACCTTTAATGCAGGAGAATTTTCTCTTCCCACTTTTGATTCACTGAATATTACTGCTGCATCTTTAAATGATGATGGATTGGATAATCATACTATACTGCTCTACTACTCAACTGCTGCTTCTAGTTTGGCTGTAACATTGATGATAGCTATTTTTATTGTTTATATGGTCTCCAGAGACAATGTTTCTTGCTCCATCTGTCTATAAGGAAAATTAAGCCCTGTATTTTCCTTTGTTGTAGTGCTTGTTTGCTTGTTACCATTACAAAGAAACGTTATTGAAAAATGCTCTTGTTACTACT (NP, B/Ann Arbor/1/66) SEQ ID NO: 38AGCAGAAGCACAGCATTTTCTTGTGAACTTCAAGTACCAACAAAAACTGAAAATCAAAATGTCCAACATGGATATTGACGGCATCAACACTGGAACAATTGACAAAACACCAGAAGAAATAACTTCCGGAACCAGTGGGGCAACCAGACCAATCATCAAGCCAGCAACCCTTGCCCCACCAAGCAATAAACGAACCCGAAACCCATCCCCAGAAAGGGCAACCACAAGCAGCGAAGCGATTGTCGGAAGGAGAACCCAAAAGAAACAAACCCCGACAGAGATAAAGAAGAGCGTCTACAATATGGTAGTGAAACTGGGTGAATTCTACAACCAGATGATGGTCAAAGCTGGACTCAACGATGACATGGAGAGAAACCTAATCCAAAATGCACATGCTGTGGAAAGAATTCTATTGGCTGCTACTGATGACAAGAAAACTGAATACCAAAAGAAAAAGAATGCCAGAGATGTCAAAGAAGGGAAAGAAGAAATAGACCACAACAAAACAGGAGGCACCTTTTATAAGATGGTAAGAGATGATAAAACCATCTACTTCAGCCCTATAAGAATTACCTTTTTAAAAGAAGAGGTGAAAACAATGTACAAGACCACCATGGGGAGTGATGGTTTCAGTGGACTAAATCACATCATGATTGGGCATTCACAGATGAACGATGTCTGTTTCCAAAGATCAAAGGCACTAAAAAGAGTTGGACTTGACCCTTCATTAATCAGTACTTTTGCAGGAAGCACACTCCCCAGAAGATCAGGTGCAACTGGTGTTGCGATCAAAGGAGGTGGAACTTTAGTGGCAGAAGCCATTCGATTTATAGGAAGAGCAATGGCAGACAGAGGGCTATTGAGAGACATCAGAGCCAAGACGGCCTATGAAAAGATTCTTCTGAATCTGAAAAACAAGTGCTCTGCGCCCCAACAAAAGGCTCTAGTTGATCAAGTGATCGGAAGTAGAAACCCAGGGATTGCAGACATAGAAGACCTAACCCTGCTTGCCCGAAGCATGGTCGTTGTCAGGCCCTCTGTAGCGAGCAAAGTGGTGCTTCCCATAAGCATTAATGCTAAAATACCTCAACTAGGGTTCAATGTTGAAGAATACTCTATGGTTGGGTATGAAGCCATGGCTCTTTATAATATGGCAACACCTGTTTCCATATTAAGAATGGGAGACGATGCAAAAGATAAATCACAATTATTCTTCATGTCTTGCTTTGGAGCTGCCTATGAAGACCAAAGAGTTTTGTCTGCACTAACCGGCACAGAATTCAAGCCTAGGTCAGCATTAAAGTGCAAGGGTTTCCACGTTCCAGCAAAGGAGCAAGTGGAAGGAATGGGGGCAGCTCTGATGTCCATCAAGCTCCAGTTTTGGGCCCCAATGACCAGATCTGGGGGGAACGAAGTAGGTGGAGACGGAGGGTCTGGTCAAATAAGTTGCAGCCCCGTGTTTGCAGTAGAGAGACCTATTGCTCTAAGCAAGCAAGCTGTAAGAAGAATGCTGTCAATGAATATTGAGGGACGTGATGCAGATGTCAAAGGAAATCTACTCAAGATGATGAATGATTCAATGGCTAAGAAAACCAATGGAAATGCTTTCATTGGGAAGAAAATGTTTCAAATATCAGACAAAAACAAAATCAATCCCGTTGATATTCCAATTAAGCAGACCATCCCCAATTTCTTCTTTGGGAGGGACACAGCAGAGGATTATGATGACCTCGATTATTAAAGCAACAAAATAGACACTATGGCTGTGACTGTTTCAGTACGTTTGGAATGTGGGTGTTTACTCTTATTGAAATAAATGTAAAAAATGCTGTTGTTTCTACT(NP, B/Ann Arbor/1/66 - alternative sequence) SEQ ID NO: 39AGCAGAAGCACAGCATTTTCTTGTGAACTTCAAGTACCAACAAAAACTGAAAATCAAAATGTCCAACATGGATATTGACGGCATCAACACTGGAACAATTGACAAAACACCAGAAGAAATAACTTCCGGAACCAGTGGGGCAACCAGACCAATCATCAAGCCAGCAACCCTTGCCCCACCAAGCAATAAACGAACCCGAAACCCATCCCCAGAAAGGGCAACCACAAGCAGCGAAGCGATTGTCGGAAGGAGAACCCAAAAGAAACAAACCCCGACAGAGATAAAGAAGAGCGTCTACAATATGGTAGTGAAACTGGGTGAATTCTACAACCAGATGATGGTCAAAGCTGGACTCAACGATGACATGGAGAGAAACCTAATCCAAAATGCACATGCTGTGGAAAGAATTCTATTGGCTGCTACTGATGACAAGAAAACTGAATACCAAAAGAAAAAGAATGCCAGAGATGTCAAAGAAGGGAAAGAAGAAATAGACCACAACAAAACAGGAGGCACCTTTTATAAGATGGTAAGAGATGATAAAACCATCTACTTCAGCCCTATAAGAATTACCTTTTTAAAAGAAGAGGTGAAAACAATGTACAAGACCACCATGGGGAGTGATGGTTTCAGTGGACTAAATCACATCATGATTGGGCATTCACAGATGAACGATGTCTGTTTCCAAAGATCAAAGGCACTAAAAAGAGTTGGACTTGACCCTTCATTAATCAGTACTTTTGCAGGAAGCACACTCCCCAGAAGATCAGGTGCAACTGGTGTTGCGATCAAAGGAGGTGGAACTTTAGTGGCAGAAGCCATTCGATTTATAGGAAGAGCAATGGCAGACAGAGGGCTATTGAGAGACATCAGAGCCAAGACGGCCTATGAAAAGATTCTTCTGAATCTGAAAAACAAGTGCTCTGCGCCCCAACAAAAGGCTCTAGTTGATCAAGTGATCGGAAGTAGAAACCCAGGGATTGCAGACATAGAAGACCTAACCCTGCTTGCCCGAAGCATGGTCGTTGTCAGGCCCTCTGTAGCGAGCAAAGTGGTGCTTCCCATAAGCATTAATGCTAAAATACCTCAACTAGGGTTCAATGTTGAAGAATACTCTATGGTTGGGTATGAAGCCATGGCTCTTTATAATATGGCAACACCTGTTTCCATATTAAGAATGGGAGACGATGCAAAAGATAAATCACAATTATTCTTCATGTCTTGCTTTGGAGCTGCCTATGAAGACCAAAGAGTTTTGTCTGCACTAACCGGCACAGAATTCAAGCCTAGGTCAGCATTAAAGTGCAAGGGTTTCCACGTTCCAGCAAAGGAGCAAGTGGAAGGAATGGGGGCAGCTCTGATGTCCATCAAGCTCCAGTTTTGGGCCCCAATGACCAGATCTGGGGGGAACGAAGTAGGTGGAGACGGAGGGTCTGGTCAAATAAGTTGCAGCCCCGTGTTTGCAGTAGAGAGACCTATTGCTCTAAGCAAGCAAGCTGTAAGAAGAATGCTGTCAATGAATATTGAGGGACGTGATGCAGATGTCAAAGGAAATCTACTCAAGATGATGAATGATTCAATGGCTAAGAAAACCAATGGAAATGCTTTCATTGGGAAGAAAATGTTTCAAATATCAGACAAAAACAAAATCAATCCCGTTGATATTCCAATTAAGCAGACCATCCCCAATTTCTTCTTTGGGAGGGACACAGCAGAGGATTATGATGACCTCGATTATTAAAGCAACAAAATAGACACTATGGCTGTGACTGTTTCAGTACGTTTGGAATGTGGGTGTTTACTCTTATTGAAATAAATGTAAAAAATGCTGTTGTTTCTACT(PB2, A/New Caledonia/20/1999) SEQ ID NO: 40ATGGAAAGAATAAAAGAGCTAAGGAATCTGATGTCACAATCTCGCACTCGCGAGATACTTACAAAAACTACTGTAGACCACATGGCCATAATCAAGAAATACACATCAGGAAGACAGGAGAAAAACCCATCACTTAGAATGAAATGGATGATGGCAATGAAATACCCAATTACAGCAGATAAAAGGATAACGGAAATGATTCCTGAAAGAAATGAGCAAGGACAGACATTATGGAGTAAAGTGAATGATGCCGGATCAGACCGAGTGATGATATCACCCCTGGCTGTGACATGGTGGAACAGAAATGGACCAGTGGCAAGTACTATTCACTATCCAAAAATCTACAAAACTTACTTTGAAAAGGTTGAAAGGTTAAAACATGGAACCTTTGGCCCTGTACACTTTAGAAACCAAGTCAAAATACGCCGAAGAGTCGACATAAATCCTGGTCATGCAGACCTCAGCGCCAAGGAGGCACAGGATGTAATTATGGAAGTTGTTTTCCCTAATGAAGTGGGAGCCAGAATACTAACATCAGAATCGCAATTAACGATAACCAAGGAGAAAAAAGAAGAACTCCAGAATTGCAAAATTTCCCCTTTGATGGTTGCATACATGTTAGAGAGGGAACTTGTCCGCAAAACGAGATTTCTCCCGGTTGCTGGTGGAACAAGCAGTGTGTACATTGAAGTTTTGCATTTAACACAGGGGACATGCTGGGAGCAGATGTACACTCCAGGTGGGGAGGTGAGGAATGATGATGTTGATCAAAGCCTAATTATTGCTGCTAGGAACATAGTGAGAAGAGCTGCAGTATCAGCAGATCCACTAGCATCTTTATTAGAAATGTGCCATAGCACACAGATTGGTGGGACAAGGATGGTGGATATTCTCAGGCAAAATCCAACAGAAGAACAAGCTGTGGATATATGCAAAGCAGCAATGGGGCTGAGAATCAGTTCATCCTTCAGTTTTGGCGGATTCACATTTAAGAGAACAAGTGGATCATCAGTCAAAAGGGAGGAAGAAGTGCTCACGGGCAATCTGCAAACATTGAAGCTAACTGTGCATGAGGGATATGAAGAGTTCACAATGGTTGGGAAAAGGGCAACAGCTATACTCAGAAAAGCAACCAGGAGATTGATTCAACTAATAGTGAGTGGAAGAGACGAACAGTCAATAGTCGAAGCAATAGTTGTAGCAATGGTATTCTCACAAGAAGATTGCATGGTAAAAGCAGTTAGAGGTGATCTGAATTTCGTTAATAGAGCGAATCAGCGGTTGAATCCCATGCATCAACTTTTGAGACATTTTCAGAAGGATGCTAAAGTACTTTTCTTAAATTGGGGAATTGAACCTATCGACAATGTGATGGGAATGATTGGGATATTACCTGATATGACTCCAAGTACCGAGATGTCAATGAGAGGAGTGAGAGTCAGCAAAATGGGTGTAGATGAATACTCCAATGCTGAAAGGGTAGTGGTGAGCATTGACCGTTTTTTGAGAGTCCGGGACCAAAGAGGAAATGTACTACTGTCTCCAGAGGAAGTCAGTGAAACACAGGGAACAGAGAAACTGACAATAACTTACTCTTCATCAATGATGTGGGAGATTAATGGCCCTGAGTCAGTGTTGATCAATACCTATCAGTGGATCATCAGAAACTGGGAGACTGTTAAAATTCAGTGGTCTCAGAACCCTACAATGCTATACAATAAAATGGAATTCGAGCCATTTCAGTCTCTAGTCCCTAAGGCCATTAGAGGCCAATACAGTGGGTTTGTTAGAACTCTATTTCAACAAATGAGGGATGTGCTTGGGACCTTTGACACAACTCAGATAATAAAACTTCTTCCCTTTGCAGCCGCTCCACCAAAGCAAAGTAGAATGCAATTCTCATCATTGACTGTGAATGTGAGGGGATCAGGAATGAGAATACTTGTAAGGGGTAATTCTCCAGTATTCAACTACAACAAGACCACTAAGAGACTCACAGTCCTCGGAAAGGATGCTGGCACTTTAACTGAAGACCCAGATGAAGGCACAGCTGGAGTGGAATCTGCTGTTCTAAGGGGATTCCTCATTCTAGGCAAAGAAGATAGAAGATATGGGCCAGCATTAAGCATCAATGAATTGAGCAACCTTGCGAAAGGGGAAAAAGCTAATGTGCTAATTGGGCAAGGGGACGTAGTGTTGGTAATGAAACGAAAACGGGACTCTAGCATACTTACTGACAGCCAGACAGCGACCAAAAGAATTCGGATGGCCATCAAT(encodes the same amino acid sequence as SEQ ID NO: 40) SEQ ID NO: 41ATGGAACGCATTAAAGAACTGCGCAACCTGATGAGCCAGAGCCGCACCCGCGAAATTCTGACCAAAACCACCGTGGATCATATGGCGATTATTAAAAAATATACCAGCGGCCGCCAGGAAAAAAACCCGAGCCTGCGCATGAAATGGATGATGGCGATGAAATATCCGATTACCGCGGATAAACGCATTACCGAAATGATTCCGGAACGCAACGAACAGGGCCAGACCCTGTGGAGCAAAGTGAACGATGCGGGCAGCGATCGCGTGATGATTAGCCCGCTGGCGGTGACCTGGTGGAACCGCAACGGCCCGGTGGCGAGCACCATTCATTATCCGAAAATTTATAAAACCTATTTTGAAAAAGTGGAACGCCTGAAACATGGCACCTTTGGCCCGGTGCATTTTCGCAACCAGGTGAAAATTCGCCGCCGCGTGGATATTAACCCGGGCCATGCGGATCTGAGCGCGAAAGAAGCGCAGGATGTGATTATGGAAGTGGTGTTTCCGAACGAAGTGGGCGCGCGCATTCTGACCAGCGAAAGCCAGCTGACCATTACCAAAGAAAAAAAAGAAGAACTGCAGAACTGCAAAATTAGCCCGCTGATGGTGGCGTATATGCTGGAACGCGAACTGGTGCGCAAAACCCGCTTTCTGCCGGTGGCGGGCGGCACCAGCAGCGTGTATATTGAAGTGCTGCATCTGACCCAGGGCACCTGCTGGGAACAGATGTATACCCCGGGCGGCGAAGTGCGCAACGATGATGTGGATCAGAGCCTGATTATTGCGGCGCGCAACATTGTGCGCCGCGCGGCGGTGAGCGCGGATCCGCTGGCGAGCCTGCTGGAAATGTGCCATAGCACCCAGATTGGCGGCACCCGCATGGTGGATATTCTGCGCCAGAACCCGACCGAAGAACAGGCGGTGGATATTTGCAAAGCGGCGATGGGCCTGCGCATTAGCAGCAGCTTTAGCTTTGGCGGCTTTACCTTTAAACGCACCAGCGGCAGCAGCGTGAAACGCGAAGAAGAAGTGCTGACCGGCAACCTGCAGACCCTGAAACTGACCGTGCATGAAGGCTATGAAGAATTTACCATGGTGGGCAAACGCGCGACCGCGATTCTGCGCAAAGCGACCCGCCGCCTGATTCAGCTGATTGTGAGCGGCCGCGATGAACAGAGCATTGTGGAAGCGATTGTGGTGGCGATGGTGTTTAGCCAGGAAGATTGCATGGTGAAAGCGGTGCGCGGCGATCTGAACTTTGTGAACCGCGCGAACCAGCGCCTGAACCCGATGCATCAGCTGCTGCGCCATTTTCAGAAAGATGCGAAAGTGCTGTTTCTGAACTGGGGCATTGAACCGATTGATAACGTGATGGGCATGATTGGCATTCTGCCGGATATGACCCCGAGCACCGAAATGAGCATGCGCGGCGTGCGCGTGAGCAAAATGGGCGTGGATGAATATAGCAACGCGGAACGCGTGGTGGTGAGCATTGATCGCTTTCTGCGCGTGCGCGATCAGCGCGGCAACGTGCTGCTGAGCCCGGAAGAAGTGAGCGAAACCCAGGGCACCGAAAAACTGACCATTACCTATAGCAGCAGCATGATGTGGGAAATTAACGGCCCGGAAAGCGTGCTGATTAACACCTATCAGTGGATTATTCGCAACTGGGAAACCGTGAAAATTCAGTGGAGCCAGAACCCGACCATGCTGTATAACAAAATGGAATTTGAACCGTTTCAGAGCCTGGTGCCGAAAGCGATTCGCGGCCAGTATAGCGGCTTTGTGCGCACCCTGTTTCAGCAGATGCGCGATGTGCTGGGCACCTTTGATACCACCCAGATTATTAAACTGCTGCCGTTTGCGGCGGCGCCGCCGAAACAGAGCCGCATGCAGTTTAGCAGCCTGACCGTGAACGTGCGCGGCAGCGGCATGCGCATTCTGGTGCGCGGCAACAGCCCGGTGTTTAACTATAACAAAACCACCAAACGCCTGACCGTGCTGGGCAAAGATGCGGGCACCCTGACCGAAGATCCGGATGAAGGCACCGCGGGCGTGGAAAGCGCGGTGCTGCGCGGCTTTCTGATTCTGGGCAAAGAAGATCGCCGCTATGGCCCGGCGCTGAGCATTAACGAACTGAGCAACCTGGCGAAAGGCGAAAAAGCGAACGTGCTGATTGGCCAGGGCGATGTGGTGCTGGTGATGAAACGCAAACGCGATAGCAGCATTCTGACCGATAGCCAGACCGCGACCAAACGCATTCGCATGGCGATTAAC (HA, BX-35) SEQ ID NO: 42MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDDETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTEDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL (NP, B/Lee/40)SEQ ID NO: 43AGCATTTTCTTGTGAGCTTCGAGCACTAATAAAACTGAAAATCAAAATGTCCAACATGGATATTGACAGTATAAATACCGGAACAATCGATAAAAAACCAGAAGAACTGACTCCCGGAACCAGTGGGGCAACCAGACCAATCATCAAGCCAGCAACCCTTGCTCCGCCAAGCAACAAACGAACCCGAAATCCATCCCCAGAAAGGACAACCACAAGCAGTGAAACCGATATCGGAAGGAAAATCCAAAAGAAACAAACCCCAACAGAGATAAAGAAGAGCGTCTACAACATGGTGGTAAAGCTGGGTGAATTCTACAACCAGATGATGGTCAAAGCTGGACTTAATGATGACATGGAAAGGAATCTAATCCAAAATGCACAAGCTGTGGAGAGAATCCTATTGGCTGCAACTGATGACAAGAAAACTGAATACCAAAAGAAAAGGAATGCCAGAGATGTCAAAGAAGGGAAGGAAGAAATAGACCACAACAAGACAGGAGGCACCTTTTATAAGATGGTAAGAGATGATAAAACCATCTACTTCAGCCCTATAAAAATTACCTTTTTAAAAGAAGAGGTGAAAACAATGTACAAGACCACCATGGGGAGTGATGGTTTCAGTGGACTAAATCACATTATGATTGGACATTCACAGATGAACGATGTCTGTTTCCAAAGATCAAAGGCACTGAAAAGGGTTGGACTTGACCCTTCATTAATCAGTACTTTTGCCGGAAGCACACTACCCAGAAGATCAGGTACAACTGGTGTTGCAATCAAAGGAGGTGGAACTTTAGTGGCAGAAGCCATTCGATTTATAGGAAGAGCAATGGCAGACAGAGGGCTACTGAGAGACATCAAGGCCAAGACAGCCTATGAAAAGATTCTTCTGAATCTGAAAAACAAGTGCTCTGCGCCCCAACAAAAGGCTCTAGTTGATCAAGTGATCGGAAGTAGGAACCCAGGGATTGCAGACATAGAAGACCTAACTCTGCTTGCCAGAAGCATGATAGTTGTCAGACCCTCTGTAGCGAGCAAAGTGGTGCTTCCCATAAGCATTTATGCTAAAATACCTCAACTAGGATTCAATATCGAAGAATACTCTATGGTTGGGTATGAAGCCATGGCTCTTTATAATATGGCAACACCTGTTTCCATATTAAGAATGGGAGATGACGCAAAAGATAAATCTCAACTATTCTTCATGTCGTGCTTCGGAGCTGCCTATGAAGATCTAAGAGTGTTATCTGCACTAACGGGCACCGAATTTAAGCCTAGATCAGCACTAAAATGCAAGGGTTTCCATGTCCCGGCTAAGGAGCAAGTAGAAGGAATGGGGGCAGCTCTGATGTCCATCAAGCTTCAGTTCTGGGCCCCAATGACCAGATCTGGAGGGAATGAAGTAAGTGGAGAAGGAGGGTCTGGTCAAATAAGTTGCAGCCCTGTGTTTGCAGTAGAAAGACCTATTGCTCTAAGCAAGCAAGCTGTAAGAAGAATGCTGTCAATGAACGTTGAAGGACGTGATGCAGATGTCAAAGGAAATCTACTCAAAATGATGAATGATTCGATGGCAAAGAAAACCAGTGGAAATGCTTTCATTGGGAAGAAAATGTTTCAAATATCAGACAAAAACAAAGTCAATCCCATTGAGATTCCAATTAAGCAGACCATCCCCAGTTTCTTCTTTGGGAGGGACACAGCAGAGGATTATGATGACCTCGATTATTAAAGCAATAAAATAGACACTATGGCTGTGACTGTTTCAGTACGTTTGGGATGTGGGTGTTTACTCTTATTGAAATAAATGTAAAA (NP, B/Ann Arbor/1/66)SEQ ID NO: 44MSNMDIDGINTGTIDKTPEEITSGTSGATRPIIKPATLAPPSNKRTRNPSPERATTSSEAIVGRRTQKKQTPTEIKKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAHAVERILLAATDDKKTEYQKKKNARDVKEGKEEIDHNKTGGTFYKMVRDDKTIYFSPIRITFLKEEVKTMYKTTMGSDGFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTLPRRSGATGVAIKGGGTLVAEAIRFIGRAMADRGLLRDIRAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRNPGIADIEDLTLLARSMVVVRPSVASKVVLPISINAKIPQLGENVEEYSMVGYEAMALYNMATPVSILRMGDDAKDKSQLFFMSCFGAAYEDQRVLSALTGTEFKPRSALKCKGFHVPAKEQVEGMGAALMSIKLQFWAPMTRSGGNEVGGDGGSGQISCSPVFAVERPIALSKQAVRRMLSMNIEGRDADVKGNLLKMMNDSMAKKTNGNAFIGKKMFQISDKNKINPVDIPIKQTIPNFFFGRDTAEDYDDLDY (NP, B/Ann Arbor/1/66) SEQ ID NO: 45MSNMDIDGINTGTIDKTPEEITSGTSGATRPIIKPATLAPPSNKRTRNPSPERAATSSEADVGRRTQKKQTPTEIKKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAHAAERILLAATDDKKTEFQKKKNARDVKEGKEEIDHNKTGGTFYKMVRDDKTIYFSPIRITFLKEEVKTMYKTTMGSDGFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTLPRRSGATGVAIKGGGTLVAEAIRFIGRAMADRGLLRDIRAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRNPGIADIEDLTLLARSMVVVRPSVASKVVLPISINAKIPQLGFNVEEYSMVGYEAMALYNMATPVSILRMGDDAKDKSQLFEMSCFGAAYEDQRVLSALTGTEFKHRSALKCKGFHVPAKEQVEGMGAALMSIKLQFWAPMTRSGGNEVGGDGGSGQISCSPVFAVERPIALSKQAVRRMLSMNIEGRDADVKGNLLKMMNDSMTKKTNGNAFIGKKMFQISDKNKTNPIEIPIKQTIPNFFFGRDTAEDYDDLDY

The invention claimed is:
 1. A method of preparing a reassortantinfluenza B virus comprising steps of: (i) introducing into a culturehost one or more expression construct(s) which encode(s) the viralsegments required to produce an influenza B virus wherein the expressionconstruct(s) encode the HA segment from a first influenza B virus andthe NP and/or PB2 segment from a second influenza B virus which is aB/Victoria/2/87-like strain; and (ii) culturing the culture host inorder to produce a reassortant influenza B virus.
 2. A method ofpreparing a reassortant influenza B virus comprising steps of: (i)introducing into a culture host one or more expression construct(s)which encode(s) the viral segments required to produce an influenza Bvirus wherein the expression construct(s) encode the HA segment from afirst influenza B virus and the NP segment from a second influenza Bvirus which is not B/Lee/40 or B/Ann Arbor/1/66 or B/Panama/45/90; and(ii) culturing the culture host in order to produce a reassortantinfluenza B virus.
 3. The method of claim 2, wherein the NP and PB2segments are from the second influenza B virus.
 4. The method of claim2, wherein the second influenza B virus is a B/Victoria/2/87-likestrain.
 5. The method of claim 1, wherein the PA, PB1, PB2, NP, NS and Msegments are from the second influenza B virus.
 6. The method of claim1, wherein the reassortant influenza B virus comprises backbone segmentsfrom two or more influenza B strains.
 7. The method of claim 6, whereinat least one backbone segment is from a B/Yamagata/16/88-like strain. 8.A method of preparing a reassortant influenza B virus comprising stepsof (i) introducing into a culture host one or more expressionconstruct(s) which encode(s) the viral segments required to produce aninfluenza B virus comprising the HA segment from a B/Yamagata/16/88-likestrain and at least one backbone segment from a B/Victoria/2/87-likestrain; and (ii) culturing the culture host in order to produce areassortant influenza B virus.
 9. The method of claim 8, wherein two,three, four, five or six backbone segments are from theB/Victoria/2/87-like strain.
 10. The method of claim 8, wherein theratio of segments from the B/Victoria/2/87-like strain and theB/Yamagata/16/88-like strain is 7:1, 6:2, 4:4, 3:5 or 1:7.
 11. A methodof preparing a reassortant influenza B virus comprising steps of (i)introducing into a culture host one or more expression construct(s)which encode(s) the viral segments required to produce an influenza Bvirus comprising viral segments from a B/Victoria/2/87-like strain and aB/Yamagata/16/88-like strain, wherein the ratio of segments from theB/Victoria/2/87-like strain and the B/Yamagata/16/88-like strain is 1:7,2:6, 3:5, 4:4, 5:3, 6:2 or 7:1; and (ii) culturing the culture host inorder to produce a reassortant influenza B virus.
 12. The method ofclaim 10, wherein the ratio is 7:1, 6:2, 4:4, 3:5 or 1:7.
 13. The methodof claim 1, wherein the B/Victoria/2/87-like strain is B/Brisbane/60/08.14. The method of claim 11, wherein the B/Yamagata/16/88-like strain isB/Panama/45/90.
 15. The method of claim 1, wherein the NP segmentencodes a protein which has at least 97%, at least 98%, at least 99%identity or 100% identity to the sequence of SEQ ID NO:
 4. 16. Themethod of claim 1, wherein the PB2 segment encodes a protein which hasat least 97%, at least 98%, at least 99% or 100% identity to thesequence of SEQ ID NO:
 3. 17. The method of claim 1, wherein the NSsegment encodes a protein which has at least 97%, at least 98%, at least99% or 100% identity with the sequence of SEQ ID NO: 35 and/or whereinthe M1 segment encodes a protein which has at least 97%, at least 98%,at least 99% or 100% identity with the sequence of SEQ ID NO:
 34. 18.The method of claim 1, wherein the reassortant influenza B viruscomprises: a) a PA protein which has at least 97% identity, at least 98%identity, at least 99% identity or 100% identity to the sequence of SEQID NO: 1; and/or b) a PB1 protein which has at least 97% identity, atleast 98% identity, at least 99% identity or 100% identity to thesequence of SEQ ID NO: 2; and/or c) a M1 protein which has at least 97%identity, at least 98%, at least 99% identity or 100% identity to thesequence of SEQ ID NO: 5; and/or d) a M2 protein which has at least 97%identity, at least 98% identity, at least 99% identity or 100% identityto the sequence of SEQ ID NO: 6; and/or e) a NS1 protein which has atleast 97% identity, at least 98%, at least 99% identity or 100% identityto the sequence of SEQ ID NO: 7; and/or f) a NS2 protein which has atleast 97% identity, at least 98%, at least 99% identity or 100% identityto the sequence of SEQ ID NO:
 8. 19. A method of preparing a reassortantinfluenza B virus comprising steps of (i) introducing into a culturehost one or more expression construct(s) which encode(s) the viralsegments required to produce an influenza B virus comprising: a) the PAsegment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12, the PB2segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15; or b) thePA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 31, the PB2segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34; or c) thePA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 31, the PB2segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15; or d) thePA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 12, the PB2segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15, or e) thePA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12, the PB2segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34; f) the PAsegment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31, the PB2segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 33, the NSsegment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or g) thePA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31, the PB2segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or h) thePA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12, the PB2segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 33, the NSsegment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or i) thePA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12, the PB2segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or j) thePA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 12, the PB2segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or k) thePA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31, the PB2segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NSsegment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34; and (ii)culturing the culture host in order to produce a reassortant influenza Bvirus.
 20. The method of claim 1, further comprising the step (iii) ofpurifying the reassortant virus obtained in step (ii).
 21. A reassortantinfluenza B virus obtainable by the method of claim
 1. 22. The method ofclaim 1, wherein the culture host is an embryonated hen egg.
 23. Themethod of claim 1, wherein the culture host is a cell.
 24. The method ofclaim 23, wherein the cell is an MDCK, Vero or PerC6 cell.
 25. Themethod of claim 23, wherein the cell grows adherently.
 26. The method ofclaim 23, wherein the cell grows in suspension.
 27. The method of claim26, wherein the MDCK cell is cell line MDCK 33016 (DSM ACC2219).
 28. Amethod of preparing a vaccine, comprising the steps of (a) preparing avirus by the method of claim 1 and (b) preparing a vaccine from thevirus.
 29. A method of preparing a vaccine, comprising the step ofpreparing a vaccine from the virus of claim
 21. 30. The method of claim28, wherein step (b) involves inactivating the virus.
 31. The method ofclaim 28, wherein the vaccine is a whole virion vaccine.
 32. The methodof claim 28, wherein the vaccine is a split virion vaccine.
 33. Themethod of claim 28, wherein the vaccine is a surface antigen vaccine.34. The method of claim 28, wherein the vaccine is a virosomal vaccine.35. The method of claim 28, wherein the vaccine contains less than 10 ngof residual host cell DNA per dose.
 36. The method of claim 28, whereinthe vaccine comprises an antigen from an influenza A strain.
 37. Themethod of claim 36, wherein the influenza A strain is of the H1, H2, H5,H7 or H9 subtype.
 38. A vaccine obtainable by the method of claim 28.39. An expression system comprising one or more expression construct(s)comprising the vRNA encoding segments of an influenza B virus of claim21.
 40. A host cell comprising the expression system of claim
 39. 41.The host cell of claim 40, wherein the host cell is a mammalian cell.42. The host cell of claim 41, wherein the host cell is an MDCK, Vero orPerC6 cell.